TWI655906B - Composition and method for controlling insects and microorganisms using pseudomonas taiwanensis - Google Patents

Composition and method for controlling insects and microorganisms using pseudomonas taiwanensis Download PDF

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TWI655906B
TWI655906B TW104118951A TW104118951A TWI655906B TW I655906 B TWI655906 B TW I655906B TW 104118951 A TW104118951 A TW 104118951A TW 104118951 A TW104118951 A TW 104118951A TW I655906 B TWI655906 B TW I655906B
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tccc
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施明哲
Ming Che Shih
劉嚞睿
Je Ruei Liu
楊玉良
Yu Liang Yang
陳文仁
Wen Jen Chen
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中央研究院
Academia Sinica
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Abstract

本文中描述使用台灣假單孢菌(Pseudomonas taiwanensis)及其培養液控制昆蟲及微生物生長之方法及組合物。 Methods and compositions for controlling insect and microbial growth using Pseudomonas taiwanensis and its culture fluid are described herein.

Description

使用台灣假單孢菌( Pseudomonas taiwanensis )控制昆蟲及微生物之組合物及方法Composition and method for controlling insects and microorganisms using Pseudomonas taiwanensis 相關申請之交叉參考 Cross-reference to related applications

本申請案主張2014年6月11日申請之美國臨時申請案第62/010,776號之優先權,其內容以全文引用的方式併入本文中。 This application claims priority from US Provisional Application No. 62 / 010,776, filed on June 11, 2014, the contents of which are incorporated herein by reference in their entirety.

使用生理、生物化學、細胞脂肪酸及16S rRNA基因序列法將台灣假單孢菌(Pseudomonas taiwanensis)(假單孢菌種TKU015)分類為新穎細菌。其自土壤分離且可在蝦殼粉末作為唯一碳及氮源之培養基上生長。台灣假單孢菌在蝦殼培養基下顯示高水準細胞外殼質酶、脫乙醯幾丁質酶及納豆激酶(nattokinase)活性。已展示,單獨自台灣假單孢菌重組TccC可導致果蠅(Drosophila)幼蟲之死亡,表明台灣假單孢菌之TccC具有其自身有毒特性。 Physiological, biochemical, cellular fatty acid and 16S rRNA gene sequence methods were used to classify Pseudomonas taiwanensis ( Pseudomonas species TKU015) as novel bacteria. It is isolated from the soil and can be grown on a medium with shrimp shell powder as the sole source of carbon and nitrogen. Pseudomonas taiwan showed high levels of cell coat enzymes, deacetylated chitinase, and nattokinase in shrimp shell culture medium. It has been shown that recombination of TccC from Pseudomonas taiwanensis alone can cause the death of Drosophila larvae, indicating that TccC of Pseudomonas taiwanensis has its own toxic properties.

本文中描述使用台灣假單孢菌控制昆蟲及微生物生長之方法及組合物。 Methods and compositions for controlling insect and microbial growth using Pseudomonas taiwanii are described herein.

在一個態樣中,本文中描述一種產生用於抑制微生物生長之組合物的方法。該方法包括在養分有限培養基中培養台灣假單孢菌菌株以獲得培養液及收集該培養液,因此產生組合物。在一個實施例中,培養基為鐵有限培養基。培養基可為補充有酪蛋白胺基酸、MgSO4及甘 油之M9基本培養基。該方法可進一步包括自培養液移除細胞以獲得無細胞上清液及收集該無細胞上清液。在一個實施例中,台灣假單孢菌菌株具有寄存編號DSM 21245。在另一實施例中,台灣假單孢菌菌株具有功能喪失rpoS突變。在一個實施例中,微生物為植物病原細菌、植物病原真菌或多重抗藥性細菌。微生物可為水稻黃單孢菌水稻致病變種(Xanthomonas oryzae pv.Oryzae)、似膠黏孢炭疽刺盤孢菌(Colletotrichum gloeosporioides)、辣椒疫黴菌(Phytophthora capsici)、稻熱病菌(Pyricularia oryzae)、立枯絲核菌(Rhizoctonia solani)、蘭花萎凋病菌(Fusarium oxysporum f sp cattleyae)、表皮葡萄球菌(Staphylococcus epidermidis)、金黃色葡萄球菌(Staphylococcus aureus)或白色念珠菌(Candida albican)。 In one aspect, described herein is a method of producing a composition for inhibiting microbial growth. The method includes culturing a Pseudomonas taiwanensis strain in a nutrient-limited medium to obtain a culture fluid and collecting the culture fluid, thereby producing a composition. In one embodiment, the medium is an iron limited medium. The medium may be a M9 basic medium supplemented with casein amino acid, MgSO 4 and glycerol. The method may further include removing cells from the culture fluid to obtain a cell-free supernatant and collecting the cell-free supernatant. In one embodiment, the Pseudomonas taiwan strain has accession number DSM 21245. In another embodiment, the Pseudomonas taiwan strain has a loss of function rpoS mutation. In one embodiment, the microorganism is a phytopathogenic bacterium, a phytopathogenic fungus, or a multi-drug resistant bacterium. The microorganisms can be Xanthomonas oryzae pv. Oryzae , Colletotrichum gloeosporioides , Phytophthora capsici , Pyricularia oryzae , Rhizoctonia solani , Fusarium oxysporum f sp cattleyae , Staphylococcus epidermidis , Staphylococcus aureus or Candida albican .

在另一態樣中,本文中描述一種用於抑制微生物生長之組合物。該組合物藉由包括在養分有限培養基中培養台灣假單孢菌菌株以獲得培養液及收集該培養液之上文所描述之方法而產生。組合物可進一步含有一或多種其他抗細菌劑、抗真菌劑或殺蟲劑。 In another aspect, a composition for inhibiting microbial growth is described herein. The composition is produced by a method described above including culturing a Pseudomonas taiwanii strain in a nutrient-limited medium to obtain a culture fluid and collecting the culture fluid. The composition may further contain one or more other antibacterial, antifungal or insecticides.

在又一態樣中,本文中描述一種抑制微生物生長之方法,其包括使微生物與上文所描述之藉由在養分有限培養基中培養台灣假單孢菌菌株產生的組合物接觸。微生物可為植物病原細菌、植物病原真菌或多重抗藥性細菌。在一個實施例中,微生物選自由以下組成之群:水稻黃單孢菌水稻致病變種、似膠黏孢炭疽刺盤孢菌、辣椒疫黴菌、稻熱病菌、立枯絲核菌、蘭花萎凋病菌、表皮葡萄球菌、金黃色葡萄球菌或白色念珠菌。 In yet another aspect, described herein is a method of inhibiting the growth of a microorganism comprising contacting the microorganism with a composition described above by culturing a Pseudomonas Taiwanese strain in a nutrient-limited medium. The microorganism may be a phytopathogenic bacterium, a phytopathogenic fungus, or a multi-drug resistant bacterium. In one embodiment, the microorganism is selected from the group consisting of Xanthomonas oryzae pv. Oryzae, Gliocladium anthracis, P. capsici, Phytophthora capsici, Rhizoctonia solani, Orchid wilting Germs, Staphylococcus epidermidis, Staphylococcus aureus or Candida albicans.

本文中亦描述一種治療或降低水稻細菌性枯葉病(rice bacterial blight)風險之方法。該方法包括將上文所描述之組合物施用至有需要之水稻植物上。 A method for treating or reducing the risk of rice bacterial blight is also described herein. The method includes applying the composition described above to a rice plant in need.

在一個態樣中,下文描述一種抑制微生物生長之方法,其包括使 微生物與具有結構Q-DSer-Lys-OHHis-aDThr-Ser-cOHOrn之經分離螢綠素(pyoverdine)接觸。Q為發色團,且微生物為植物病原細菌、植物病原真菌或多重抗藥性細菌。在一個實施例中,微生物選自由以下組成之群:水稻黃單孢菌水稻致病變種、似膠黏孢炭疽刺盤孢菌、辣椒疫黴菌、稻熱病菌、立枯絲核菌、蘭花萎凋病菌、表皮葡萄球菌、金黃色葡萄球菌或白色念珠菌。 In one aspect, a method of inhibiting microbial growth is described below, which includes The microorganism is contacted with an isolated pyoverdine having the structure Q-DSer-Lys-OHHis-aDThr-Ser-cOHOrn. Q is a chromophore, and the microorganism is a plant pathogenic bacteria, a plant pathogenic fungus, or a multi-drug resistant bacteria. In one embodiment, the microorganism is selected from the group consisting of Xanthomonas oryzae pv. Oryzae, Gliocladium anthracis, P. capsici, Phytophthora capsici, Rhizoctonia solani, Orchid wither Germs, Staphylococcus epidermidis, Staphylococcus aureus or Candida albicans.

在另一態樣中,本文中描述一種抑制昆蟲生長之方法,其包括使昆蟲與含有台灣假單孢菌菌株、台灣假單孢菌細胞溶解物或台灣假單孢菌TccC多肽之組合物接觸。昆蟲為鱗翅目(Lepidopteran)物種。在一個實施例中,昆蟲為小菜蛾(Plutella xylostella)、甜菜夜蛾(Spodoptera exigua)或粉紋夜蛾(Trichoplusia ni)。在一個實施例中,細胞溶解物為全細胞溶解物或可溶性溶解物。台灣假單孢菌菌株可在富含養分之培養基中經培養,且細胞溶解物可自富含養分之培養基中培養之台灣假單孢菌菌株獲得。 In another aspect, a method for inhibiting the growth of an insect is described herein, comprising contacting an insect with a composition comprising a Pseudomonas taiwan strain, a Pseudomonas taiwan cell lysate, or a Pseudomonas taiwanensis TccC polypeptide . Insects Lepidoptera (Lepidopteran) species. In one embodiment, the insect is a Plutella xylostella , a Spodoptera exigua , or a Trichoplusia ni . In one embodiment, the cell lysate is a whole cell lysate or a soluble lysate. Pseudomonas taiwan strains can be cultured in nutrient-rich media, and cell lysates can be obtained from Pseudomonas taiwan strains cultured in nutrient-rich media.

一或多個實施例之細節闡述於以下隨附圖式及實施方式中。實施例之其他特徵、目標及優點將自實施方式及圖式及申請專利範圍顯而易見。 Details of one or more embodiments are set forth in the accompanying drawings and embodiments below. Other features, objectives, and advantages of the embodiments will be apparent from the embodiments and drawings and the scope of patent applications.

圖1為展示螢綠素結構及ESI軌道阱質譜中之特徵離子的一組示意圖及圖表。 Figure 1 is a set of schematic diagrams and diagrams showing the fluorescein structure and characteristic ions in the ESI orbital trap mass spectrum.

圖2為(a)台灣假單孢菌與(b)綠膿假單孢菌(P.aeroginsa)POA1之螢綠素基因座之示意性比較。 Figure 2 is a schematic comparison of the luciferin locus of (a) Pseudomonas taiwan and (b) P. aeroginsa POA1.

圖3為一組(a)展示成熟螢綠素之亞細胞定位之圖表及(b)台灣假單孢菌中之螢綠素分泌路徑的示意性圖式。 Figure 3 is a set of (a) a diagram showing the subcellular localization of mature chlorophyll and (b) a schematic diagram of the chlorophyll secretion pathway in Pseudomonas taiwanensis.

圖4為一組(A)展示與內部對照16S rRNA基因(白色三角形)之TccC表現量相比,在台灣假單孢菌之不同生長階段(灰色條形圖)期間之 TccC表現量的圖表。在OD600(黑色圓)處量測台灣假單孢菌之生長曲線,及(B)展示經台灣假單孢菌處理之小菜蛾幼蟲之相片。 Figure 4 is a group (A) showing the TccC expression levels of the 16S rRNA gene (white triangle) of the internal control during different growth stages (gray bar graph) of Pseudomonas taiwanensis. TccC performance chart. The growth curve of Pseudomonas taiwanensis was measured at OD600 (black circle), and (B) a photograph showing Plutella xylostella larvae treated with Pseudomonas taiwanensis.

圖5為一組展示台灣假單孢菌及各種細胞部分對草地黏蟲(Spodoptera frugiperda)Sf9昆蟲細胞之毒性的圖表。(A)台灣假單孢菌野生型及△tccC(MOI=1000)及來源於台灣假單孢菌之(B)細胞溶解物、(C)可溶性溶解物及(D)不溶性溶解物之蛋白部分(10μg/ml)感染後之Sf9細胞的存活率。96孔盤中之每孔含有5000個Sf9細胞。結果在台灣假單孢菌感染或蛋白處理72h後,藉由XTT增殖分析獲得。 FIG. 5 is a set of graphs showing the toxicity of Pseudomonas taiwanii and various cell parts to Spodoptera frugiperda Sf9 insect cells. (A) Protein fractions of Pseudomonas taiwan wild type and △ tccC (MOI = 1000) and (B) cell lysate, (C) soluble lysate and (D) insoluble lysate derived from Pseudomonas taiwanensis (10 μg / ml) survival rate of Sf9 cells after infection. Each well in a 96-well plate contains 5000 Sf9 cells. The results were obtained by XTT proliferation analysis 72 hours after Pseudomonas infection or protein treatment in Taiwan.

圖6為自台灣假單孢菌培養液分離不同蛋白部分之程序示意圖。 FIG. 6 is a schematic diagram of a procedure for isolating different protein fractions from a Pseudomonas culture medium in Taiwan.

本文中描述一種產生抑制微生物生長之組合物的方法。該方法包括在養分有限培養基中培養台灣假單孢菌菌株以獲得培養液。收集培養液以獲得該組合物。 Described herein is a method of producing a composition that inhibits microbial growth. The method includes culturing a Pseudomonas taiwanensis strain in a nutrient-limited medium to obtain a culture solution. The culture broth was collected to obtain the composition.

養分有限培養基可為不具有鐵源之培養基,例如鐵有限培養基。舉例而言,培養基可為M9培養基,其可補充有其他養分(例如,酪蛋白胺基酸、MgSO4及甘油)。菌株可在鐵有限培養基中在25℃至37℃下培養1至6天。培養基可含有一定低量鐵,只要該量足夠低以允許產生有效針對靶向微生物之培養液。 The nutrient-limited medium may be a medium without an iron source, such as an iron-limited medium. For example, the medium may be an M9 medium, which may be supplemented with other nutrients (eg, casein amino acid, MgSO 4, and glycerol). The strain can be cultured in an iron-limited medium at 25 ° C to 37 ° C for 1 to 6 days. The medium may contain a certain low amount of iron as long as the amount is low enough to allow the production of a culture medium effective against the targeted microorganism.

所得培養液可用作抑制微生物生長之組合物。視情況,細胞可自培養液移除以獲得無細胞上清液,其可隨後用作該組合物。 The obtained culture solution can be used as a composition for inhibiting the growth of microorganisms. Optionally, the cells can be removed from the culture to obtain a cell-free supernatant, which can then be used as the composition.

本文中亦描述一種使用具有結構Q-DSer-Lys-OHHis-aDThr-Ser-cOHOrn之經分離螢綠素抑制微生物生長之方法,其中Q為發色團。此類螢綠素可藉由在鐵有限培養基中培養台灣假單孢菌菌株及分離由此產生之螢綠素來獲得。 Also described herein is a method for inhibiting microbial growth using isolated chlorophyll with the structure Q-DSer-Lys-OHHis-aDThr-Ser-cOHOrn, where Q is a chromophore. Such fluorescein can be obtained by culturing a Pseudomonas taiwanensis strain in an iron-limited medium and isolating the fluorescein produced thereby.

此外,本發明包括一種抑制昆蟲生長之方法。該方法包括使昆蟲與含有台灣假單孢菌菌株、台灣假單孢菌細胞溶解物或台灣假單孢菌 TccC多肽之組合物接觸。 In addition, the invention includes a method for inhibiting the growth of insects. The method includes contacting an insect with a strain containing Pseudomonas taiwanii, Pseudomonas taiwan cell lysate, or Pseudomonas taiwanensis A composition of TccC polypeptide is contacted.

細胞溶解物可為全細胞溶解物或可溶性溶解物。細胞溶解物可藉由在富含養分之培養基(例如,LB培養基或1/2 TSB培養基)中培養台灣假單孢菌菌株,使細胞分裂及隨後收集細胞溶解物來獲得。細胞溶解物可經過濾、離心或以其他方式處理以分離可溶性溶解物及不溶性溶解物。舉例而言,可使用圖6中展示之程序。 The cell lysate may be a whole cell lysate or a soluble lysate. Cell lysates can be obtained by culturing Pseudomonas taiwanii strains in a nutrient-rich medium (eg, LB medium or 1/2 TSB medium), dividing the cells, and then collecting the cell lysates. Cell lysates can be filtered, centrifuged, or otherwise processed to separate soluble and insoluble lysates. For example, the procedure shown in Figure 6 can be used.

台灣假單孢菌TccC多肽可使用此項技術中已知之技術獲得。下方展示台灣假單孢菌TccC之核酸序列(SEQ ID NO:1)及胺基酸序列(SEQ ID NO:2)。 Pseudomonas taiwanensis TccC polypeptides can be obtained using techniques known in the art. The nucleic acid sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of Pseudomonas taiwanensis TccC are shown below.

(SEQ ID NO:1) (SEQ ID NO: 1)

(SEQ ID NO:2) (SEQ ID NO: 2)

一或多種額外殺蟲劑、抗真菌劑或抗細菌劑可添加至藉由本文中描述之方法產生或本文中描述之方法中使用之組合物中。該等試劑包括(但不限於)鏈黴環素(鏈黴素硫酸鹽及四環素,例如10%)、克枯爛(Tecloftalam)(例如10%)、撲殺熱(Probenazole)(例如6%或10%)、培丹 鹽酸鹽(Cartap hydrochloride)、芳族烴、胍、二甲醯亞胺、2-胺基嘧啶、有機磷、苯并咪唑、甲醯胺、固醇生物合成抑制劑、抗卵菌綱、嗜毬果傘素(strobilurin)、苯胺基嘧啶、苯基吡咯苯甲醯胺、喹諾酮及Bt殺蟲毒素。 One or more additional insecticides, antifungals or antibacterials can be added to the composition produced by the method described herein or used in the method described herein. Such reagents include, but are not limited to, streptomycin (streptomycin sulfate and tetracycline, such as 10%), Tecloftalam (such as 10%), Probenazole (such as 6% or 10) %), Petan Hydrochloride (Cartap hydrochloride), aromatic hydrocarbons, guanidine, dimethylformimine, 2-aminopyrimidine, organophosphorus, benzimidazole, formamidine, sterol biosynthesis inhibitors, anti-oomycetes, Strobilurin, aniline pyrimidine, phenylpyrrole benzamidine, quinolone and Bt insecticide.

其他試劑,諸如失活成分(例如,防腐劑、載劑、溶劑及染料)亦可包括於組合物中。 Other agents, such as inactivated ingredients (e.g., preservatives, vehicles, solvents, and dyes) can also be included in the composition.

用於本文中描述之方法中之台灣假單孢菌菌株可為具有寄存編號DSM 21245之菌株。菌株亦可為具有功能喪失rpoS突變之突變菌株。此類菌株可使用此項技術中已知之重組及/或基因技術產生。台灣假單孢菌rpoS之核酸序列(SEQ ID NO:3)及胺基酸序列(SEQ ID NO:4)展示如下: (SEQ ID NO:3) The Pseudomonas taiwanii strain used in the methods described herein may be a strain with deposit number DSM 21245. The strain may also be a mutant strain having a loss of function rpoS mutation. Such strains can be produced using recombinant and / or genetic techniques known in the art. The nucleic acid sequence (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 4) of Pseudomonas taiwanensis rpoS are shown below: (SEQ ID NO: 3)

(SEQ ID NO:4) (SEQ ID NO: 4)

上文所描述之組合物及方法中之任一者可用於抑制各種昆蟲及微生物(例如,植物病原細菌、植物病原真菌或多重抗藥性細菌)之生長。其亦可用於治療由昆蟲及微生物引起的疾病或減少其風險,例如由水稻黃單孢菌水稻致病變種引起的水稻細菌性枯葉病。舉例而言,組合物可投與(例如,噴灑至)經感染或未感染標靶(例如,水稻植物)。 Any of the compositions and methods described above can be used to inhibit the growth of various insects and microorganisms (eg, plant pathogenic bacteria, plant pathogenic fungi, or multi-drug resistant bacteria). It can also be used to treat or reduce the risk of diseases caused by insects and microorganisms, such as rice bacterial leaf blight caused by Xanthomonas oryzae pv. Oryzae. For example, the composition can be administered (e.g., sprayed onto) an infected or uninfected target (e.g., a rice plant).

微生物包括(但不限於)水稻黃單孢菌水稻致病變種(Xoo)、水稻黃 單孢菌條斑致病變種(Xanthomonas oryzae pv.oryzicola,Xoc)、多犯性植物炭疽病菌(Colletotrichum acutatum)、龍舌蘭炭疽病菌(Colletotrichum agaves)、埃爾氏炭疽病菌(Colletotrichum alcornii)、花生炭疽病菌(Colletotrichum arachidis)、巴氏炭疽病菌(Colletotrichum baltimorense)、辣椒炭疽病菌(Colletotrichum capsici)、尾狀核炭疽病菌(Colletotrichum caudatum)、穀類炭疽病菌(Colletotrichum cereal)、馬鈴薯炭疽病菌(Colletotrichum coccodes)、水葫蘆炭疽病菌(Colletotrichum crassipes)、蔥蘭(Colletotrichum dematium)、代氏炭疽病菌(Colletotrichum derridis)、菸草炭疽病菌(Colletotrichum destructivum)、草莓炭疽病菌(Colletotrichum fragariae)、芒果炭疽病菌(Colletotrichum gloeosporioides)、棉黑斑炭疽病菌(Colletotrichum gossypii)、禾生炭疽病菌(Colletotrichum graminicola)、菜心炭疽病菌(Colletotrichum higginsianum)、咖啡炭疽病菌(Colletotrichum kahawae)、菜豆炭疽病菌(Colletotrichum lindemuthianum)、亞麻炭疽病菌(Colletotrichum lini)、曼氏炭疽病菌(Colletotrichum mangenotii)、香蕉炭疽病菌(Colletotrichum musae)、黑刺盤孢菌(Colletotrichum nigrum)、黃瓜炭疽病菌(Colletotrichum orbiculare)、豌豆炭疽病菌(Colletotrichum pisi)、賽氏炭疽病菌(Colletotrichum somersetense)、柿樹炭疽病菌(Colletotrichum sublineolum)、常春藤炭疽菌(Colletotrichum trichellum)、三葉草炭疽病菌(Colletotrichum trifolii)、大豆炭疽病菌(Colletotrichum truncatum)、葡萄炭疽病菌(Colletotrichum viniferum)、結縷草炭疽病菌(Colletotrichum zoysiae)、貝殼杉類疫黴菌(Phytophthora taxon Agathis)、擬赤楊屬疫黴菌(Phytophthora alni)、棉鈴疫病黴菌(Phytophthora boehmeriae)、簇囊疫黴菌(Phytophthora botryose)、甘藍蚜疫黴菌(Phytophthora brassicae)、惡疫黴菌(Phytophthora cactorum)、木豆疫黴菌(Phytophthora cajani)、 受栗疫黴菌(Phytophthora cambivora)、辣椒疫黴菌(Phytophthora capsici)、樟疫黴菌(Phytophthora cinnamomi)、柑橘疫病菌(Phytophthora citricola)、柑橘褐腐疫黴(Phytophthora citrophthora)、克萊氏疫黴菌(Phytophthora clandestine)、芋疫黴菌(Phytophthora colocasiae)、隱地疫黴菌(Phytophthora cryptogea)、甜瓜疫黴菌(Phytophthora drechsleri)、迪瓦阿克曼疫黴菌(Phytophthora diwan ackerman)、馬鈴薯疫黴緋腐病菌(Phytophthora erythroseptica)、草莓疫黴菌(Phytophthora fragariae)、草莓紅心疫黴菌(Phytophthora fragariae var.rubi)、雙子疫黴菌(Phytophthora Gemini)、格氏疫黴菌(Phytophthora glovera)、節水黴狀疫黴菌(Phytophthora gonapodyides)、橡膠樹疫黴菌(Phytophthora heveae)、冬生疫黴菌(Phytophthora hibernalis)、腐植疫黴菌(Phytophthora humicola)、海氏疫黴菌(Phytophthora hydropathical)、灌溉疫黴菌(Phytophthora irrigate)、越橘疫黴菌(Phytophthora idaei)、節桿疫黴菌(Phytophthora ilicis)、致病疫黴(Phytophthora infestans)、膨脹疫黴菌(Phytophthora inflate)、紅薯疫黴菌(Phytophthora ipomoeae)、依氏疫黴菌(Phytophthora iranica)、桂氏疫黴菌(Phytophthora katsurae)、雪松疫黴菌(Phytophthora lateralis)、苜蓿疫黴菌(Phytophthora medicaginis)、梅根氏疫黴菌(Phytophthora megakarya)、大豆疫病菌(Phytophthora megasperma)、瓜類疫黴菌(Phytophthora melonis)、紫茉莉疫黴菌(Phytophthora mirabilis)、蘭花疫黴菌(Phytophthora multivesiculata)、山菅蘭疫黴菌(Phytophthora nemorosa)、菸草疫黴菌(Phytophthora nicotianae)、潘尼氏疫黴菌(Phytophthora PaniaKara)、棕櫚疫黴菌(Phytophthora palmivora)、菜豆疫黴菌(Phytophthora phaseoli)、松花疫黴菌(Phytophthora pini)、韭蔥疫黴菌(Phytophthora porri)、夏櫟疫黴菌(Phytophthora plurivora)、櫻草疫黴菌(Phytophthora primulae)、 偽丁香疫黴疫黴菌(Phytophthora pseudosyringae)、花旗松疫黴菌(Phytophthora pseudotsugae)、櫟疫黴菌(Phytophthora quercina)、櫟樹猝死疫黴菌(Phytophthora ramorum)、甜橙疫黴菌(Phytophthora sinensis)、大豆疫黴菌(Phytophthora sojae)、丁香疫黴(Phytophthora syringae)、觸角豆螺疫黴菌(Phytophthora tentaculata)、三葉草疫黴菌(Phytophthora trifolii)、豇豆疫黴菌(Phytophthora vignae)、鈍角梨孢(Pyricularia angulate)、紅樹梨孢(Pyricularia apiculata)、沙茴香梨孢(Pyricularia borealis)、布氏梨孢(Pyricularia buloloensis)、醋栗梨孢(Pyricularia caffra)、美人蕉生孢梨孢(Pyricularia cannae)、堪尼氏梨孢(Pyricularia cannicola)、陰地莎草梨孢(Pyricularia caricis)、鴨蹠草梨孢(Pyricularia commelinicola)、閉鞘薑梨孢(Pyricularia costi)、科斯蒂納梨孢(Pyricularia costina)、薑黃素梨孢(Pyricularia curcumae)、七島蘭梨孢(Pyricularia cyperi)、馬薄荷梨孢(Pyricularia didyma)、大指草梨孢(Pyricularia digitariae)、代氏梨孢(Pyricularia distorta)、天山梨孢(Pyricularia dubiosa)、艾比氏梨孢(Pyricularia ebbelsii)、禾長蠕梨孢(Pyricularia echinochloae)、千金子梨孢(Pyricularia euphorbiae)、羽瑚梨孢(Pyricularia fusispora)、格魯氏梨孢(Pyricularia globbae)、灰梨孢(Pyricularia grisea)、古氏梨孢(Pyricularia guarumaicola)、棕扇尾鶯梨孢(Pyricularia juncicola)、庫氏梨孢(Pyricularia kookicola)、月桂梨孢(Pyricularia lauri)、李氏禾梨孢(Pyricularia leersiae)、馬蹄包梨孢(Pyricularia longispora)、羅氏梨孢(Pyricularia lourinae)、地楊梅梨孢(Pyricularia luzulae)、西花梨孢(Pyricularia occidentalis)、椰子樹梨孢(Pyricularia oncosperma)、稻熱病菌、水生黍梨孢(Pyricularia panici-paludosi)、寄生霜梨抱(Pyricularia parasitica)、狼尾草梨孢(Pyricularia penniseti)、佩氏梨孢(Pyricularia peruamazonica)、似梨形梨孢(Pyricularia pyricularioides)、拉包爾志賀氏梨孢(Pyricularia rabaulensis)、虎尾蘭梨孢(Pyricularia sansevieriae)、文字梨孢(Pyricularia scripta)、粟梨孢(Pyricularia setariae)、星砂梨孢(Pyricularia sphaerulata)、淹沒梨孢(Pyricularia submerse)、亞斯格瑪梨孢(Pyricularia subsigmoidea)、梵氏梨孢(Pyricularia vandalurensis)、魚腥藻梨孢(Pyricularia variabilis)、生楊樹梨孢(Pyricularia whetzelii)、薑擬梨孢(Pyricularia zingiberis)、角斑梨孢(Pyricularia zizaniicola)、合歡木絲核菌(Rhizoctonia bataticola)、胡蘿菔絲核菌(Rhizoctonia carotae)、禾穀絲核菌(Rhizoctonia cerealis)、紫紋羽絲核菌(Rhizoctonia crocorum)、草莓絲核菌(Rhizoctonia fragariae)、斑葉蘭絲核菌(Rhizoctonia goodyerae-repentis)、豆狀絲核(Rhizoctonia leguminicola)、水稻絲核菌(Rhizoctoni oryzae)、小麥角菌根菌(Ceratorhiza ramicola)、玉蜀黍絲核菌(Rhizoctonia zeae)、椰棗尖鐮孢菌(Fusarium oxysporum f.sp.albedinis)、蘆筍莖尖鐮孢菌(Fusarium oxysporum f.sp.asparagi)、甘薯尖鐮孢菌(Fusarium oxysporum f.sp.batatas)、甜菜尖鐮孢菌(Fusarium oxysporum f.sp.betae)、大麻尖鐮孢菌(Fusarium oxysporum f.sp.cannabis)、洋蔥尖鐮孢菌(Fusarium oxysporum f.sp.cepae)、鷹嘴豆尖鐮孢菌(Fusarium oxysporum f.sp.ciceris)、柑橘尖鐮孢菌(Fusarium oxysporum f.sp.citri)、咖啡尖鐮孢菌(Fusarium oxysporum f.sp.coffea)、香蕉尖鐮孢菌(Fusarium oxysporum f.sp.cubense)、荔枝尖鐮孢菌(Fusarium oxysporum f.sp.cyclaminis)、草尖鐮孢菌(Fusarium oxysporum f.sp.herbemontis)、瞿麥尖鐮孢菌(Fusarium oxysporum f.sp.dianthi)、萵苣尖鐮孢菌(Fusarium oxysporum f.sp.lactucae)、乳香黃連木尖鐮孢菌(Fusarium oxysporum f.sp.lentis)、亞麻尖鐮孢菌(Fusarium oxysporum f.sp.lini)、番茄尖鐮孢菌(Fusarium oxysporum f.sp.lycopersici)、苜蓿尖鐮孢菌(Fusarium oxysporum f.sp.medicaginis)、瓜類尖鐮孢菌(Fusarium oxysporum f.sp. melonis)、菸草尖鐮孢菌(Fusarium oxysporum f.sp.nicotianae)、西瓜尖鐮孢菌(Fusarium oxysporum f.sp.niveum)、棕櫚尖鐮孢菌(Fusarium oxysporum f.sp.palmarum)、西番蓮尖鐮孢菌(Fusarium oxysporum f.sp.passiflorae)、菜豆尖鐮孢菌(Fusarium oxysporum f.sp.phaseoli)、豌豆尖鐮孢菌(Fusarium oxysporum f.sp.pisi)、番茄根尖鐮孢菌(Fusarium oxysporum f.sp.radicis-lycopersici)、蓖麻蠶尖鐮孢菌(Fusarium oxysporum f.sp.ricini)、獨腳金草尖鐮孢菌(Fusarium oxysporum f.sp.strigae)、晚香玉尖鐮孢菌(Fusarium oxysporum f.sp.tuberosi)、鬱金香尖鐮孢菌(Fusarium oxysporum f.sp.tulipae)、棉花尖鐮孢菌(Fusarium oxysporum f.sp.vasinfectum)、阿爾萊葡萄球菌(Staphylococcus arlettae)、阿根提葡萄球菌(Staphylococcus agnetis)、金黃色葡萄球菌、耳葡萄球菌(Staphylococcus auricularis)、頭葡萄球菌(Staphylococcus capitis)、山羊葡萄球菌(Staphylococcus caprae)、肉葡萄球菌(Staphylococcus carnosus)、臘腸葡萄球菌(Staphylococcus caseolyticus)、色葡萄球菌(Staphylococcus chromogenes)、科氏葡萄球菌(Staphylococcus cohnii)、康氏葡萄球菌(Staphylococcus condiment)、海豚葡萄球菌(Staphylococcus delphini)、德氏葡萄球菌(Staphylococcus devriesei)、表皮葡萄球菌、馬胃葡萄球菌(Staphylococcus equorum)、貓葡萄球菌(Staphylococcus felis)、福氏葡萄球菌(Staphylococcus fleurettii)、雞葡萄球菌(Staphylococcus gallinarum)、溶血性葡萄球菌(Staphylococcus haemolyticus)、人類葡萄球菌(Staphylococcus hominis)、豬葡萄球菌(Staphylococcus hyicus)、中間葡萄球菌(Staphylococcus intermedius)、克氏葡萄球菌(Staphylococcus kloosii)、李氏葡萄球菌(Staphylococcus leei)、緩慢葡萄球菌(Staphylococcus lentus)、里昂葡萄球菌(Staphylococcus lugdunensis)、水獺葡萄球菌(Staphylococcus lutrae)、馬賽葡萄球菌(Staphylococcus massiliensis)、 田鼠葡萄球菌(Staphylococcus microti)、蠅葡萄球菌(Staphylococcus muscae)、尼泊爾葡萄球菌(Staphylococcus nepalensis)、巴氏葡萄球菌(Staphylococcus pasteuri)、佩氏葡萄球菌(Staphylococcus pettenkoferi)、魚葡萄球菌(Staphylococcus piscifermentans)、假中間葡萄球菌(Staphylococcus pseudintermedius)、假里昂葡萄球菌(Staphylococcus pseudolugdunensis)、普氏葡萄球菌(Staphylococcus pulvereri)、豬鼻葡萄球菌(Staphylococcus rostri)、解糖葡萄球菌(Staphylococcus saccharolyticus)、腐生性葡萄球菌(Staphylococcus saprophyticus)、斯蓋氏葡萄球菌(Staphylococcus schleiferi)、松鼠葡萄球菌(Staphylococcus sciuri)、金猿葡萄球菌(Staphylococcus simiae)、模仿葡萄球菌(Staphylococcus simulans)、斯氏葡萄球菌(Staphylococcus stepanovicii)、琥珀葡萄球菌(Staphylococcus succinus)、小牛葡萄球菌(Staphylococcus vitulinus)、瓦氏葡萄球菌(Staphylococcus warneri)、木糖葡萄球菌(Staphylococcus xylosus)、白色念珠菌、阿氏念珠菌(Candida ascalaphidarum)、安氏念珠菌(Candida amphixiae)、南極念珠菌(Candida Antarctica)、青葙念珠菌(Candida argentea)、大西洋念珠菌(Candida atlantica)、大氣念珠菌(Candida atmosphaerica)、布拉特念珠菌(Candida blattae)、鳳梨念珠菌(Candida bromeliacearum)、卡氏念珠菌(Candida carpophila)、卡發氏念珠菌(Candida carvajalis)、天牛念珠菌(Candida cerambycidarum)、堪氏念珠菌(Candida chauliodes)、紫堇念珠菌(Candida corydalis)、多西氏念珠菌(Candida dosseyi)、都氏念珠菌(Candida dubliniensis)、埃氏念珠菌(Candida ergatensis)、果實念珠菌(Candida fructus)、光滑念珠菌(Candida glabrata)、醱酵念珠菌(Candida fermentati)、高里氏念珠菌(Candida guilliermondii)、哈氏念珠菌屬(Candida haemulonii)、昆蟲念珠菌(Candida insectamens)、因氏念珠菌(Candida insectorum)、媒介念珠菌(Candida intermedia)、傑夫 氏念珠菌(Candida jeffresii)、克菲爾氏念珠菌(Candida kefyr)、科氏念珠菌(Candida keroseneae)、克魯斯氏念珠菌(Candida krusei)、葡萄牙念珠菌(Candida lusitaniae)、里氏念珠菌屬(Candida lyxosophila)、麥芽糖念珠菌(Candida maltose)、海生念珠菌(Candida marina)、酵母菌念珠菌(Candida membranifaciens)、米氏念珠菌(Candida milleri)、橄欖念珠菌(Candida oleophila)、歐氏念珠菌(Candida oregonensis)、近平滑念珠菌(Candida parapsilosis)、橡樹念珠菌(Candida quercitrusa)、皺褶念珠菌(Candida rugosa)、米酒念珠菌(Candida sake)、謝氏念珠菌(Candida shehatea)、忒氏念珠菌(Candida temnochilae)、留蘭香念珠菌(Candida tenuis)、司氏念珠菌(Candida theae)、托氏念珠菌(Candida tolerans)、熱帶念珠菌(Candida tropicalis)、土屋念珠菌(Candida tsuchiyae)、賽諾氏念珠菌(Candida sinolaborantium)、黴菌念珠菌(Candida sojae)、亞哈氏念珠菌(Candida subhashii)、維斯氏念珠菌(Candida viswanathii)、高蛋白念珠菌(Candida utilis)及俄氏念珠菌(Candida ubatubensis)。 Microorganisms include (but are not limited to) X. oryzae (Xoo), Xanthomonas oryzae pv.oryzicola (Xoc), or Colletotrichum acutatum . agave anthracnose (Colletotrichum agaves), El apos anthracnose (Colletotrichum alcornii), peanut anthracnose (Colletotrichum arachidis), Pap anthracnose (Colletotrichum baltimorense), pepper anthracnose (Colletotrichum capsici), caudate nucleus anthrax pathogen (Colletotrichum caudatum), cereals anthracnose fungus (Colletotrichum cereal), potato anthracnose fungus (Colletotrichum coccodes), water hyacinth anthracnose fungus (Colletotrichum crassipes), onions Portland (Colletotrichum dematium), Daphne anthracnose fungus (Colletotrichum derridis), tobacco anthrax bacteria (Colletotrichum destructivum), strawberry anthracnose (Colletotrichum fragariae), Colletotrichum gloeosporioides (Colletotrichum gloeosporioides), cotton spots anthracnose (Colletotrichum gossypii), graminicola anthracnose (Colletotrichum graminicola), (Colletotrichum higginsianum) anthracnose fungus, coffee anthracnose fungus (Colletotrichum kahawae), bean anthracnose (Colletotrichum lindemuthianum) bacteria, flax anthracnose fungus (Colletotrichum lini), Man anthracnose fungus (Colletotrichum mangenotii), banana anthracnose fungus (Colletotrichum musae), black Colletotrichum nigrum , Colletotrichum orbiculare , Colletotrichum pisi , Colletotrichum somersetense , Colletotrichum sublineolum , Colletotrichum anthracis trilum ), Colletotrichum trifolii , Colletotrichum truncatum , Colletotrichum viniferum , Colletotrichum zoysiae , Phytophthora taxon Agathis Phytophthora alni , Phytophthora boehmeriae , Phytophthora botryose , Phytophtho ra brassicae ), Phytophthora cactorum , Phytophthora cajani , Phytophthora cambivora , Phytophthora capsici , Phytophthora cinnamomi , Citrus Phytophthora citricola ), Phytophthora citrophthora , Phytophthora clandestine , Phytophthora colocasiae , Phytophthora cryptogea , Phytophthora oryza Phytophthora diwan ackerman , Phytophthora erythroseptica , Phytophthora fragariae , Phytophthora fragariae var.rubi , Gemini ), Grignard Phytophthora (Phytophthora glovera), saving mold shaped Phytophthora (Phytophthora gonapodyides), rubber tree Phytophthora (Phytophthora heveae), winter Phytophthora fungus (Phytophthora hibernalis), humic Phytophthora (Phytophthora humicola), Hay P. Fungus (Phytophthora hydropathical), irrigation Phytophthora (Phytophthora irrigate), cowberry Phytophthora (Phytophthora idaei), segmented rod Phytophthora (Phytophthora ilicis), Phytophthora infestans (Phytophthora infestans), Phytophthora expansion (Phytophthora inflate), sweet potato Phytophthora ipomoeae , Phytophthora iranica , Phytophthora katsurae , Phytophthora lateralis , Phytophthora medicagitopis , Megayar ), soybean blight (Phytophthora megasperma), melons Phytophthora (Phytophthora melonis), Mirabilis Phytophthora (Phytophthora mirabilis), orchids Phytophthora (Phytophthora multivesiculata), Dianella Phytophthora (Phytophthora nemorosa), tobacco Phytophthora (Phytophthora nicotianae), Penny's Phytophthora (Phytophthora PaniaKara), palm Phytophthora (Phytophthora palmivora), bean Phytophthora (Phytophthora phaseoli), Songhua Phytophthora (Phytophthora pini), leek Phytophthora (Phytophthora porri), Quercus Phytophthora (Phytophthora plurivora), primrose Phytophthora (Phytophthora primulae), clove pseudo Phytophthora infestans (Phytophthora pseudosyringae), Douglas fir Phytophthora (Phytophthora pseudotsugae), Quercus Phytophthora (Phytophthora quercina), sudden oak Phytophthora (Phytophthora ramorum), orange Phytophthora (Phytophthora sinensis), Phytophthora sojae (Phytophthora sojae), clove Phytophthora (Phytophthora syringae), spiro antennae bean Phytophthora (Phytophthora tentaculata), clover Phytophthora (Phytophthora trifolii), cowpea Phytophthora Mold ( Phytophthora vignae ), Pyricularia angulate , Pyricularia apiculata , Pyricularia borealis , Pyricularia buloloensis , Pyricularia caffra , Pyricularia cannae , Pyricularia cannicola , Pyricularia caricis , Pyricularia commelinicola , Pyricularia costi Pyricularia costina ), Curcumin Pyricularia (Pyricularia curcumae), Seven Islands blue Pyricularia (Pyricularia cyperi), horse mint Pyricularia (Pyricularia didyma), thumb grass Pyricularia (Pyricularia digitariae), Daphne Pyricularia (Pyricularia distorta), Tianshan pear spore (Pyricularia dubiosa), Abby's Pyricularia (Pyricularia ebbelsii), Wo Helminthosporium Pyricularia (Pyricularia echinochloae), daughter of the sub Pyricularia (Pyricularia euphorbiae), Yu Hu Pyricularia (Pyricularia fusispora), Turkoglu's Pyricularia ( Pyricularia globbae ), Pyricularia grisea , Pyricularia guarumaicola , Pyricularia juncicola , Pyricularia kookicola , Pyricularia lauri , plum Pyricularia leersiae , Pyricularia longispora , Pyricularia lourinae , Pyricularia luzulae , Pyricularia occidentalis , Pyricularia oncosper ), Pyricularia oryzae , Pyricularia panici-paludosi , Pyricu laria parasitica), Pennisetum Pyricularia (Pyricularia penniseti), Petri Pyricularia (Pyricularia peruamazonica), like pear Pyricularia (Pyricularia pyricularioides), Rabaul Shigella Pyricularia (Pyricularia rabaulensis), Sansevieria Pyricularia ( Pyricularia sansevieriae ), Pyricularia scripta , Pyricularia setariae , Pyricularia sphaerulata , Pyricularia submerse , Pyricularia subsigmoidea , Sanskrit Pyricularia vandalurensis , Pyricularia variabilis , Pyricularia whetzelii , Pyricularia zingiberis , Pyricularia zizaniicola , Rhizoctonia solani Rhizoctonia bataticola , Rhizoctonia carotae , Rhizoctonia cerealis , Rhizoctonia crocorum , Rhizoctonia fragariae , Rhizoctonia fragaria Rhizoctonia goodyerae-repentis , Rhizoctonia leguminicola , rice Rhizoctoni oryzae , Ceratorhiza ramicola , Rhizoctonia zeae , Fusarium oxysporum f.sp.albedinis , Fusarium oxysporum f.sp.albedinis Fusarium oxysporum f.sp.asparagi ), Fusarium oxysporum f.sp.batatas , Fusarium oxysporum f.sp.betae , Fusarium oxysporum f.sp .cannabis ), Fusarium oxysporum f.sp.cepae , Fusarium oxysporum f.sp.ciceris , Fusarium oxysporum f.sp.citri , Fusarium oxysporum f.sp.citri , Fusarium oxysporum f.sp.coffea , Fusarium oxysporum f.sp.cubense , Fusarium oxysporum f.sp.cyclaminis , Fusarium oxysporum (Fusarium oxysporum f.sp.herbemontis), Dianthus Fusarium oxysporum (Fusarium oxysporum f.sp.dianthi), lettuce Fusarium oxysporum (Fusarium oxysporum f.sp.lactucae) bacteria, mastic Fusarium oxysporum (of Fusarium oxysporum f.sp.lentis), flax Fusarium oxysporum (Fusar ium oxysporum f.sp.lini ), Fusarium oxysporum f.sp.lycopersici , Fusarium oxysporum f.sp.medicaginis , Fusarium oxysporum f. sp. melonis), tobacco Fusarium oxysporum (Fusarium oxysporum f.sp.nicotianae), watermelon Fusarium oxysporum (Fusarium oxysporum f.sp.niveum) bacteria, palm Fusarium oxysporum (Fusarium oxysporum f.sp.palmarum), Fusarium oxysporum f.sp.passiflorae , Fusarium oxysporum f.sp.phaseoli , Fusarium oxysporum f.sp.pisi , tomato root tip Fusarium oxysporum f.sp.radicis-lycopersici , Fusarium oxysporum f.sp.ricini , Fusarium oxysporum f.sp.strigae , Fusarium oxysporum f.sp.strigae , tuberose Fusarium oxysporum (Fusarium oxysporum f.sp.tuberosi), tulips Fusarium oxysporum (F usarium oxysporum f.sp.tulipae) bacteria, cotton Fusarium oxysporum (Fusarium oxysporum f.sp.vasinfectum) bacteria, E Erlai Staphylococcus arlettae , Staphyl ococcus agnetis ), Staphylococcus aureus, Staphylococcus auricularis , Staphylococcus capitis , Staphylococcus caprae , Staphylococcus carnosus , Staphylococcus caseolyticus staphylococcus Staphylococcus chromogenes , Staphylococcus cohnii , Staphylococcus condiment , Staphylococcus delphini , Staphylococcus devriesei , Staphylococcus epidermidis ( Staphylococcus equorum ), Staphylococcus felis , Staphylococcus fleurettii , Staphylococcus gallinarum , Staphylococcus haemolyticus , Staphylococcus hominis , Staphylococcus hominis ( Staphylococcus hyicus ), Staphylococcus intermedius , Staphylococcus kloosii , Staphylococ cus leei), slow staphylococci (Staphylococcus lentus), Staphylococcus Lyon (Staphylococcus lugdunensis), Staphylococcus otter (Staphylococcus lutrae), Marseille staphylococci (Staphylococcus massiliensis), voles staphylococci (Staphylococcus microti), flies staphylococci (Staphylococcus muscae ), Staphylococcus nepalensis , Staphylococcus pasteuri , Staphylococcus pettenkoferi , Staphylococcus piscifermentans , Staphylococcus pseudintermedius , Pseudolyon Staphylococcus pseudolugdunensis ), Staphylococcus pulvereri , Staphylococcus rostri , Staphylococcus saccharolyticus , Staphylococcus saprophyticus , Staphylococcus sclerococcus Staphylococcus sciuri , Staphylococcus simiae , Staphylococcus mimic lococcus simulans ), Staphylococcus stepanovicii , Staphylococcus succinus , Staphylococcus vitulinus , Staphylococcus warneri , Staphylococcus white xylos Bacteria, Candida ascalaphidarum , Candida amphixiae , Candida Antarctica , Candida argentea , Candida atlantica , Candida atmosphaerica ), Candida blattae , Candida bromeliacearum , Candida carpophila , Candida carvajalis , Candida cerambycidarum , Candida Candida chauliodes , Candida corydalis , Candida dosseyi , Candida dubliniensis , Candida ergatensis , Candida fructus ), Candi da glabrata ), Candida fermentati , Candida guilliermondii , Candida haemulonii , Candida insectamens , Candida insectorum , Candida insectorum , media candidiasis (Candida intermedia), Jeff's Candida (Candida jeffresii), kefir's Candida (Candida kefyr), Coriolis candidiasis (Candida keroseneae), Cruise's Candida (Candida krusei), Portugal candidiasis (Candida lusitaniae), Richter Candida species (Candida lyxosophila), maltose candidiasis (Candida maltose), marine candidiasis (Candida marina), yeast Candida (Candida membranifaciens), Michaelis candidiasis (Candida milleri ), Candida oleophila , Candida oregonensis , Candida parapsilosis , Candida quercitrusa , Candida rugosa , Candida sake), Xie candidiasis (Candida shehatea), Listeria candidiasis (Candida temnochilae), stay Hong candidiasis (Candida tenuis), Stormer candidiasis (Candida theae), prop's Candida (Candida tolerans), C. tropicalis (Candida tropicalis), Tsuchiya candidiasis (Candida tsuchiyae), P. knowlesi race Candida (Candida sinolaborantium ), Candida sojae , Candida subhashii , Candida viswanathii , Candida utilis , and Candida ubatubensis .

昆蟲包括鱗翅目種之彼等昆蟲,例如小菜蛾、甜菜夜蛾及粉紋夜蛾。 Insects include other insects of the Lepidoptera species, such as Plutella xylostella, Spodoptera exigua, and Spodoptera litura.

以下特定實例僅解釋為例示性的,且不以任何方式限制本發明之其餘部分。無需進一步詳細描述,咸信熟習此項技術者可基於本文中之描述最大程度地利用本發明。本文中所引用之所有公開案均以全文引用的方式併入本文中。 The following specific examples are to be construed as illustrative only and do not limit the rest of the invention in any way. Without further detailed description, those skilled in the art can make the best use of the present invention based on the description herein. All publications cited herein are incorporated herein by reference in their entirety.

實例1:來自台灣假單孢菌之螢綠素之VI型分泌系統介導之分泌物抑制水稻病原體水稻黃單孢菌水稻致病變種生長 Example 1 : Secretions mediated by the type VI secretion system of chlorophyll from Pseudomonas in Taiwan inhibit the growth of rice pathogen Xanthomonas oryzae pv. Oryzae

由水稻黃單孢菌水稻致病變種(Xoo)引起的水稻細菌性枯葉病為世界上最具破壞性之水稻疾病之一。吾人展示台灣假單孢菌顯示針對Xoo之較強拮抗活性。使用MALDI-TOF成像質譜分析(MALDI-IMS), 吾人識別由可抑制Xoo生長之台灣假單孢菌分泌之螢綠素。經由台灣假單孢菌之Tn5突變誘發,吾人展示編碼VI型分泌系統(T6SS)及螢綠素生物合成及成熟之組分之基因中的突變導致針對Xoo之毒性減小。吾人之資料證實螢綠素可經由T6SS分泌入培養基中,從而抑制Xoo生長。因此,吾人之資料不同於報導藉由T6SS之效應子遞送需要供體與受體之間的實體接觸之研究。 Bacterial rice leaf disease caused by X. oryzae rice pathogenic species ( Xoo ) is one of the most destructive rice diseases in the world. I show that Pseudomonas taiwanensis shows strong antagonistic activity against Xoo . Using MALDI-TOF imaging mass spectrometry (MALDI-IMS), we identified chlorophyll secreted by Pseudomonas taiwanii , which inhibits the growth of Xoo . Induced by the Tn5 mutation of Pseudomonas taiwanensis, we show that mutations in genes encoding the type VI secretion system (T6SS) and chlorophyll biosynthetic and mature components result in reduced toxicity to Xoo . Our data confirm that chlorophyll can be secreted into the culture medium via T6SS, thereby inhibiting Xoo growth. Therefore, our information differs from studies that report that the effector delivery by T6SS requires physical contact between donor and recipient.

藉由全基因組突變誘發之相關基因的抗Xoo活性及識別Anti- Xoo activity and recognition of related genes induced by genome-wide mutations

吾人測試若干假單孢菌物種以探索針對Xoo之潛在生物控制劑。當與富含養分之培養基(LB及½TSB)相比在鐵有限培養基上生長時,台灣假單孢菌顯示最高抗Xoo活性。在此等培養基中,台灣假單孢菌具有類似生長速率。與台灣假單孢菌對比,丁香假單孢菌(P.syringae)DC3000不展現針對Xoo之毒性。 I tested several Pseudomonas species to explore potential biological control agents against Xoo . Pseudomonas taiwanii showed the highest anti-Xoo activity when grown on iron-limited media compared to nutrient-rich media (LB and ½TSB). In these media, Pseudomonas taiwanii has a similar growth rate. Compared with Pseudomonas taiwanensis, P. syringae DC3000 does not exhibit toxicity against Xoo .

為了識別影響針對Xoo之台灣假單孢菌拮抗活性之因子,吾人產生台灣假單孢菌之Tn5誘變庫且篩選具有減弱的針對Xoo之拮抗活性的突變體。使用TAIL-PCR確定突變體之插入位點。在此等突變體中,吾人發現4個突變體之生長不受影響且其顯示減弱的針對Xoo之拮抗活性。此等突變體在編碼T6SS(clpV)、螢綠素合成酶(pvdL)、螢綠素移位及成熟(pvdE)及調節子(rpoS)之基因中具有插入位點。 In order to identify the factors that affect the Pseudomonas taiwanese antagonistic activity against Xoo , we generated a Tn5 mutagenesis library of Pseudomonas taiwanensis and screened mutants with reduced antagonistic activity against Xoo . The insertion site of the mutant was determined using TAIL-PCR. Among these mutants, we found that the growth of 4 mutants was unaffected and that they showed reduced antagonistic activity against Xoo . These mutants have insertion sites in genes encoding T6SS ( clpV ), chlorophyll synthase ( pvdL ), chlorophyll translocation and maturation ( pvdE ), and regulators ( rpoS ).

ATP酶ClpV為T6SS裝置之重要組分且有助於VipA/VipB小管重塑。參見Bonemann等人,EMBO J 28,315-325(2009)。PvdL為與螢綠素發色團之生物合成有關的肽合成酶。參見Mossialos等人,Mol Microbiol 45,1673-1685(2002)。PvdE為與螢綠素前驅物移位至周質有關之細胞膜蛋白。參見Ravel及Cornelis,Trends Microbiol 11,195-200(2003)。在鐵有限LP培養液中,自4h(停滯期)至72h(死亡期)未偵測到野生型(WT)與突變菌株(△clpV及△pvdL)之間的顯著生長差異。 The ATPase ClpV is an important component of the T6SS device and contributes to VipA / VipB tubule remodeling. See Bonemann et al., EMBO J 28,315-325 (2009). PvdL is a peptide synthetase involved in the biosynthesis of the chlorophyll chromophore. See Mossialos et al., Mol Microbiol 45, 1673-1685 (2002). PvdE is a cell membrane protein associated with the translocation of luciferin precursors into the periplasm. See Ravel and Cornelis, Trends Microbiol 11, 195-200 (2003). In iron-limited LP medium, no significant growth difference between wild type (WT) and mutant strains (ΔclpV and ΔpvdL) was detected from 4h (stagnant period) to 72h (dead period).

在拮抗分析中,野生型台灣假單抱菌之整個培養物或無細胞培養 物上清液展示針對Xoo之實質性毒性。相比而言,△clpV之整個培養物或無細胞上清液展示與WT相比之低毒性。△pvdL與△pvdE突變體均不展現對Xoo之毒性。 In an antagonistic assay, whole cultures or cell-free culture supernatants of wild-type Pseudomonas taiwanii showed substantial toxicity against Xoo . In contrast, whole cultures or cell-free supernatants of ΔclpV exhibited lower toxicity compared to WT. Neither the pvdL nor the pvdE mutants exhibited toxicity to Xoo .

藉由T6SS表徵台灣假單孢菌螢綠素針對Xoo之毒性及其分泌物Toxicity and secretion of Pseudomonas fluorescens against Xoo by T6SS

吾人使用MALDI-IMS在瓊脂盤表面上研究來自台灣假單孢菌之野生型及突變體之分泌代謝物,從而在瓊脂盤表面上研究來自台灣假單孢菌之分泌代謝物及化合物。在盤中偵測到野生型台灣假單孢菌之m/z 1044信號,而△clpVm/z 1044之含量遠低於野生型中之含量。然而,在△pvdL及△pvdE周圍未偵測到m/z 1044化合物,表明m/z 1044一種螢綠素類似物。 I used MALDI-IMS to study the secreted metabolites of wild type and mutants from Pseudomonas taiwan on the surface of agar plates, and to study the secreted metabolites and compounds from Pseudomonas taiwan on the surface of agar plates. The m / z 1044 signal of Pseudomonas taiwanensis was detected in the dish, and the content of m / z 1044 in Δ clpV was much lower than that in the wild type. However, no m / z 1044 compound was detected around ΔpvdL and ΔpvdE , indicating that m / z 1044 is a luciferin analog.

使用Cu-瓊脂糖凝膠管柱純化螢綠素,且藉由MADLI-IMS檢查。在HPLC分析中,藉由UV偵測器監測在400nm處具有最強吸光率之螢光螢綠素。來自△clpV突變體培養物之上清液具有比野生型低的螢綠素濃度。使用LC-MS之定量展示野生型中之螢綠素含量比△clpV突變體中約高2倍。吾人未在△pvdL及△pvdE突變體之培養物上清液中偵測到螢綠素。 The luciferin was purified using a Cu-sepharose column and checked by MADLI-IMS. In HPLC analysis, fluorescein with the strongest absorbance at 400 nm was monitored by a UV detector. Supernatants from delta clpV mutant cultures have a lower chlorophyll concentration than the wild type. Quantitative display using LC-MS showed that the chlorophyll content in the wild type was about 2 times higher than that in the ΔclpV mutant. We did not detect luciferin in the culture supernatants of the ΔpvdL and ΔpvdE mutants.

若干研究已在綠膿假單孢菌(Pseudomonas aeruginosa)、霍亂弧菌(Vibrio cholera)及泰國伯克霍爾德氏菌(Burkholderia thailandensis)中表徵T6SS介導之抗細菌活性。此等研究展示經由細胞-細胞接觸抗細菌效應子蛋白經由T6SS直接注入標靶細胞中。在吾人之研究中,野生型台灣假單孢菌之培養物上清液顯示比T6SS突變體△clpV較高的針對Xoo之毒性,表明抗Xoo化合物之T6SS介導之分泌不需要細胞-細胞接觸。 Several studies have characterized T6SS-mediated antibacterial activity in Pseudomonas aeruginosa , Vibrio cholera , and Burkholderia thailandensis . These studies have shown that the antibacterial effector protein is directly injected into target cells via T6SS via cell-to-cell contact. In our study, culture supernatants of wild-type Pseudomonas taiwanii showed higher toxicity against Xoo than T6SS mutant △ clpV , indicating that T6SS-mediated secretion of anti- Xoo compounds does not require cell-cell contact .

為了驗證clp V突變在台灣假單孢菌中影響T6SS活性,執行兩個實驗。首先,西方墨點分析(western blot analysis)用於定量無細胞培養物上清液中之VgrG蛋白含量,該蛋白質為針對T6SS活性之生物標記。結 果展示VgrG可在野生型及補充clpV之菌株△clpV/clpV的無細胞培養物上清液中偵測到。相比而言,未能在clpV突變體之培養物上清液中偵測到VgrG之顯著含量。結果亦展示細胞溶解物中VgrG之含量在野生型、△clpV及△clpV/clpV之間類似。RNA聚合酶α次單元RpoA用作內參考物。此等結果證實clp V突變體在T6SS功能方面有缺陷,且將野生型clp V基因引入此突變體中可恢復T6SS功能。此等結果表明,在台灣假單孢菌中,T6SS藉由將螢綠素分泌入培養基中與抗Xoo活性有關。其次,吾人藉由將clp V基因之野生型複本引入△clp V突變體中執行補充測試。在MALDI-IMS分析中,引入野生型clpV恢復螢綠素在培養物上清液中之分泌含量。資料表明△clpV突變體中螢綠素分泌減少由clpV基因座中之突變引起。 To verify that clp V mutation affects T6SS activity in Pseudomonas taiwanii, two experiments were performed. First, western blot analysis was used to quantify the VgrG protein content in the cell-free culture supernatant, which is a biomarker for T6SS activity. The results showed that VgrG was detectable in the cell-free culture supernatant of the wild-type and clpV -supplemented strain Δ clpV / clpV . In contrast, no significant amount of VgrG was detected in the culture supernatant of the clpV mutant. The results also showed that the VgrG content in the cell lysates was similar between wild-type, ΔclpV, and ΔclpV / clpV . The RNA polymerase alpha subunit RpoA was used as an internal reference. These results confirm that the clp V mutant is defective in T6SS function, and that the introduction of a wild-type clp V gene into this mutant can restore T6SS function. These results indicate that in Pseudomonas taiwanii, T6SS is associated with anti- Xoo activity by secreting luciferin into the culture medium. Second, we performed a supplemental test by introducing a wild-type copy of the clp V gene into a delta clp V mutant. In MALDI-IMS analysis, wild-type clpV was introduced to restore the secreted content of luciferin in the culture supernatant. The data indicate that reduced chlorophyll secretion in △ clpV mutants is caused by mutations in the clpV locus.

為了證實來自台灣假單孢菌之螢綠素的抗Xoo活性,藉由CAS瓊脂盤分析測試不同濃度之經純化螢綠素。在CAS瓊脂盤上,在1.2mg及1.5mg螢綠素反應時快速偵測CAS反應速率,其量測藉由螢綠素自CAS染料之鐵移除。證實螢綠素活性之後,測試針對Xoo之細胞生長抑制(IC50)及致死劑量(LD50)。針對Xoo之螢綠素之IC50為約2.035mg/ml(R2=0.9946)。LD50為約1.98mg/ml(R2=0.9775)。IC50及LD50資料展示螢綠素具有抗Xoo活性。 To confirm the anti-Xoo activity of chlorophyll from Pseudomonas taiwanii, different concentrations of purified chlorophyll were tested by CAS agar plate analysis. On a CAS agar plate, the CAS reaction rate was quickly detected when 1.2mg and 1.5mg fluorescein reacted, and its measurement was removed from the iron of the CAS dye by fluorescein. After confirming the activity of firefly chlorophyll, Xoo tested for inhibition of cell growth (IC 50) and lethal dose (LD 50). The IC 50 for Xoo 's chlorophyll is about 2.035 mg / ml (R 2 = 0.9946). The LD 50 was about 1.98 mg / ml (R 2 = 0.9775). IC 50 and LD 50 data show that chlorophyll has anti- Xoo activity.

為了進一步闡明螢綠素在台灣假單孢菌針對Xoo之拮抗活性中之作用,富含鐵之培養基用於檢查螢綠素活性。當額外鐵施用於含Xoo盤時,台灣假單孢菌之培養液展示毒性中劑量依賴性減小。在較高鐵濃度(300μM、600μM及1000μM FeCl3)下,台灣假單孢菌幾乎對Xoo無拮抗活性。與對照(僅1/2 TSB)相比,台灣假單孢菌生長不受添加鐵影響。總之,結果表明螢綠素針對Xoo之拮抗活性經由鐵競爭機制。吾人提出,當環境中鐵量有限時,台灣假單孢菌藉由將螢綠素分泌至螯合鐵中有效競爭鐵,且經由PvdRT-OpmQ溶解螢綠素-鐵錯合物,導致 Xoo之遲延生長。然而,在較高鐵濃度下,由台灣假單孢菌分泌之螢綠素不足以吸收所有可獲得的鐵,損害其抗Xoo活性。 To further clarify the role of luciferin in the anti-Xoo antagonism activity of Pseudomonas taiwanensis , an iron-rich medium was used to check luciferin activity. When additional iron was applied to Xoo- containing dishes, the culture solution of Pseudomonas taiwanii showed a dose-dependent reduction in toxicity. At higher iron concentrations (300 μM, 600 μM, and 1000 μM FeCl 3 ), Pseudomonas taiwan has almost no antagonistic activity against Xoo . Compared with the control (only 1/2 TSB), Pseudomonas taiwanii growth was not affected by the addition of iron. In summary, the results indicate that the antagonistic activity of chlorophyll against Xoo is via an iron competition mechanism. I have proposed that when the amount of iron in the environment is limited, Pseudomonas taiwan competes for iron effectively by secreting fluorescein into chelated iron, and dissolves the fluorescein-iron complex through PvdRT-OpmQ, resulting in Xoo 's Delayed growth. However, at higher iron concentrations, the chlorophyll secreted by Pseudomonas taiwan is insufficient to absorb all available iron and impair its anti- Xoo activity.

識別台灣假單孢菌中螢綠素之結構、基因座及功能Identify the structure, locus and function of chlorophyll in Pseudomonas taiwanensis

使經純化螢綠素(m/z 1044)經受串聯質譜分析以識別胺基酸之一級結構及順序。參見圖1。胺基酸序列之順序對應於NRPS腺苷醯化域特異性(Ser-Lys及Thr-Ser-OH-Orn)之預測子。此來自台灣假單孢菌之螢綠素與來自螢光假單孢菌(P.fluorescens)9AW及惡臭假單孢菌(P.putida)9BW之螢綠素一致。參見Budzikiewicz等人,Z.Naturforsch.Sect.C 52,721(1997)。 The purified luciferin ( m / z 1044) was subjected to tandem mass spectrometry to identify the amino acid primary structure and order. See Figure 1. The sequence of the amino acid sequence corresponds to the predictors of NRPS adenylate domain specificity (Ser-Lys and Thr-Ser-OH-Orn). This chlorophyll from Pseudomonas in Taiwan is consistent with fluorescein from P.fluorescens 9AW and P. putida 9BW. See Budzikiewicz et al., Z. Naturforsch. Sect. C 52,721 (1997).

螢綠素含有具有不同胺基酸組成之可變肽側鏈及保守螢光發色團。螢綠素側鏈之肽在螢光假單孢菌物種中高度可變。螢綠素之生物合成及輸送已在綠膿假單孢菌PAO1中經廣泛研究。大部分螢綠素生物合成及輸送基因在台灣假單孢菌與綠膿假單孢菌(P.aeruginosa)PAO1中形成叢群,而pvdL基因定位於兩個物種之獨立叢群中。參見圖2。pvdL基因與所有假單孢菌中螢綠素前驅物之保守螢光發色團的合成有關。pvdLpvdJpvdD之同源物與螢綠素之肽主鏈之生物合成有關。螢綠素前驅物藉由PvdE轉移入細胞質之周質空間中,PvdE為內部膜輸送體,且隨後藉由PvdA、Q、N、M、O及P加工為成熟螢綠素。PvdA為催化Om羥基化之膜結合L-鳥胺酸(Om)Nδ-加氧酶。成熟之後,螢光螢綠素分泌入細胞外環境中。PvdMpvdNpvdOpvdApvdE基因在台灣假單孢菌中叢群在一起。 Luciferin contains variable peptide side chains with different amino acid compositions and a conserved fluorescent chromophore. Peptides of the fluorescein side chain are highly variable in Pseudomonas fluorescens species. The biosynthesis and transport of luciferin have been extensively studied in P. aeruginosa PAO1. Most chlorophyll biosynthesis and transport genes form clusters in Pseudomonas and P. aruginosa PAO1, and the pvdL gene is located in a separate cluster of two species. See Figure 2. The pvdL gene is involved in the synthesis of a conserved fluorescent chromophore of the chlorophyll precursor in all Pseudomonas. The homologues of pvdL , pvdJ and pvdD are related to the biosynthesis of the peptide backbone of luciferin. The luciferin precursor is transferred into the periplasmic space of the cytoplasm by PvdE, which is an internal membrane transporter, and is subsequently processed into mature chlorophyll by PvdA, Q, N, M, O, and P. PvdA is a membrane-bound L-guanine (Om) N δ -oxygenase that catalyzes the hydroxylation of Om. After maturation, fluorescein is secreted into the extracellular environment. The PvdM , pvdN , pvdO , pvdA and pvdE genes are clustered together in Pseudomonas taiwanensis .

syrP基因(其編碼螢綠素生物合成調節性蛋白)存在於台灣假單孢菌中pvdl之下游。相比而言,syrP基因定位於丁香假單孢菌DC3000、惡臭假單孢菌KT2440及螢光假單孢菌Pf0-1中pvd基因叢群之中間。SyrP蛋白在Asp之羥基化中起作用,且與丁香黴素(syringomycin E)產生有關,其藉由NRPS合成。然而,syrP之同源未在綠膿假單孢菌PAO1 中識別。 The syrP gene, which encodes a luciferin biosynthetic regulatory protein, exists downstream of pvdl in Pseudomonas taiwanensis . In contrast, the syrP gene is located in the middle of the pvd gene cluster in P. syringae DC3000, P. putida KT2440, and P. fluorescens Pf0-1. The SyrP protein plays a role in the hydroxylation of Asp and is related to the production of syringomycin E, which is synthesized by NRPS. However, the homology of syrP was not recognized in P. aeruginosa PAO1.

表徵T6SS在螢綠素分泌中之作用Characterize the role of T6SS in luciferin secretion

為了表徵T6SS在螢綠素分泌中之作用,吾人使用IMS定量在clp V突變體及野生型之培養物中分泌之螢綠素。在鐵有限條件下,在時程實驗中培育12h之後發現螢綠素(m/z 1044.44)在台灣假單孢菌群落周圍。在16h時,瓊脂盤表面上clpV突變體中螢綠素之量遠低於野生型之量。然而,亦在clpV突變體之瓊脂盤上偵測到螢綠素。此由於螢綠素在長時間培育後積聚於培養基中,甚至在野生型及clpV突變體中。瓊脂盤之橫截面IMS展示,36h培育之後由clp V突變體分泌之螢綠素之量低於野生型之量。另一方面,IMS資料展示螢綠素不受Xoo刺激。 To characterize the role of T6SS in chlorophyll secretion, we used IMS to quantify chlorophyll secreted in clp V mutants and wild-type cultures. Under iron-limited conditions, luciferin ( m / z 1044.44) was found around the Pseudomonas community in Taiwan after 12 hours of incubation in time course experiments. At 16h, the amount of chlorophyll in the clpV mutant on the surface of the agar plate was much lower than that of the wild type. However, luciferin was also detected on agar plates of clpV mutants. This is due to the accumulation of luciferin in the medium after long-term incubation, even in wild-type and clpV mutants. The cross section IMS of the agar plate showed that the amount of chlorophyll secreted by the clp V mutant after 36h incubation was lower than that of the wild type. On the other hand, IMS data shows that chlorophyll is not stimulated by Xoo .

為了進一步評估T6SS與螢綠素分泌有關,吾人定量野生型及具有缺陷性抗Xoo活性之三種突變體的細胞外上清液、周質及細胞質中之成熟螢綠素(螢光螢綠素)。參見圖3a。在野生型與△clpV中,螢綠素之量在細胞外上清液中最高,在周質中低得多且未在細胞質中能偵測到。 參見圖3a。當詳細比較時,△clpV突變體之細胞外上清液中發現的螢綠素比野生型之螢綠素少(左圖,圖3a)。相比而言,與野生型相比,△clpV突變體在周質中積聚略微更多成熟螢綠素(中間圖,圖3a)。未在△pvdL△pvdE之亞細胞部分中之任一者中偵測到顯著數量之螢綠素。資料亦證實,PvdL及PvdE與螢綠素之生物合成及成熟有關。綜合而言,此等結果表明△clpV突變不影響細胞內螢綠素產生,但的確影響螢綠素自周質移位至培養基。 In order to further evaluate the relationship between T6SS and chlorophyll secretion, we quantified mature chlorophyll (fluorescein) in the extracellular supernatant, periplasm, and cytoplasm of three mutants of wild-type and three mutants with defective anti- Xoo activity. . See Figure 3a. In wild-type and ΔclpV , the amount of fluorescein was highest in the extracellular supernatant, much lower in the periplasm and not detectable in the cytoplasm. See Figure 3a. When compared in detail, less luciferin was found in the extracellular supernatant of the ΔclpV mutant than the wild type (left, Figure 3a). In contrast, the ΔclpV mutant accumulates slightly more mature chlorophyll in the periplasm compared to the wild type (middle panel, Figure 3a). No significant amount of chlorophyll was detected in either of the subcellular fractions of ΔpvdL and ΔpvdE . The data also confirmed that PvdL and PvdE are related to the biosynthesis and maturation of luciferin. Taken together, these results indicate that the ΔclpV mutation does not affect intracellular chlorophyll production, but does affect the translocation of luciferin from the periplasm to the culture medium.

圖3b中展示在台灣假單孢菌中之螢綠素輸送之示意圖。 Figure 3b shows a schematic diagram of luciferin transport in Pseudomonas taiwanensis.

藉由RpoS之螢綠素表現之陰性對照Negative control with luciferin performance by RpoS

生長停滯期δ因子(RpoS)為全域應激反應調節子。吾人識別在鐵有限培養基中展現增加的螢綠素產生之rpoS台灣假單孢菌突變體。3天燒瓶培育之後,與野生型中之淡綠色相比,rpoS突變菌株之培育在鐵有 限培養基下展現深綠色,且rpoS突變體不影響細胞生長。此可能因為螢光顏料螢綠素之量在培養基中積聚而展現深綠色。在拮抗分析中,與野生型相比,rpoS突變體展示對Xoo之較大抑制區。IMS資料展示,rpoS突變體比野生型分泌更多螢綠素。螢綠素之定量展示,rpoS突變體在鐵有限上清液中產生比野生型高2-3倍之螢綠素濃度。此等結果表明台灣假單孢菌中螢綠素產生受RpoS之負調節。 Growth stagnation factor δ (RpoS) is a global stress response regulator. We identified a rpoS Taiwan pseudomonas mutant that exhibited increased chlorophyll production in iron-limited media. After the 3-day flask incubation, compared to the pale green color in the wild type, the cultivation of the rpoS mutant strain showed a dark green color in the iron-limited medium, and the rpoS mutant did not affect cell growth. This may be due to the accumulation of the fluorescent pigment fluorescein in the medium and exhibit a dark green color. In the antagonism assay, the rpoS mutant displayed a larger area of inhibition of Xoo compared to the wild type. IMS data show that rpoS mutants secrete more chlorophyll than wild type. Quantification of luciferin showed that rpoS mutants produced 2-3 times higher fluorescein concentrations in iron-limited supernatants than wild type. These results indicate that chlorophyll production in Pseudomonas taiwan is negatively regulated by RpoS.

材料及方法Materials and methods (1)微生物及拮抗分析 (1) Microbial and antagonist analysis

自土壤分離台灣假單孢菌新種CMST(=BCRC17751T=DSM 21245T),且使用表現型及分子分類學方法表徵。參見Wang,L.T.等人,International Journal of Systematic and Evolutionary Microbiology 60,2094-2098(2009)。在台灣台中,自水稻細菌性枯葉病分離水稻黃單孢菌水稻致病變種(Xoo)XF89b菌株。由國立台灣大學之植物生物學研究所(Institute of Plant Biology,National Taiwan University)之Laurent Zimmerli提供丁香假單孢菌變種番茄(Pst DC3000)。 A new species of Pseudomonas CMS T (= BCRC17751 T = DSM 21245 T ) was isolated from soil and characterized by phenotypic and molecular taxonomic methods. See Wang, LT et al., International Journal of Systematic and Evolutionary Microbiology 60, 2094-2098 (2009). In Taichung, Taiwan, Xanthomonas oryzae pv. Oryzae ( Xoo ) XF89b strain was isolated from rice bacterial blight. A variety of P. syringae tomato ( Pst DC3000) was provided by Laurent Zimmerli of the Institute of Plant Biology, National Taiwan University.

在28℃下,在1/2胰蛋白酶大豆瓊脂(TSB)瓊脂盤(BD Biosciences)上測試台灣假單孢菌針對水稻細菌性枯葉病水稻黃單孢菌水稻致病變種(Xoo)之拮抗活性。使台灣假單孢菌預先培養物在鐵有限培養基(補充有1%酪蛋白胺基酸、1mM MgSO4及0.5%甘油之M9基本培養基)中生長,且在28℃及200rpm下將其培育入含100ml培養基之500ml燒瓶中持續24h。Xoo預先培養物在28℃下在1/2 TSB培養基中生長3天。混合Xoo與熔融1/2瓊脂培養基,隨後傾倒入空盤中。對於生物分析,將台灣假單孢菌(109CFU/ml)或過濾(0.22μm)上清液注入Xoo混合LB瓊脂盤之孔中直至抑制區經表徵。 Antagonistic activity of Pseudomonas taiwanensis against Xanthomonas oryzae pv. Oryzae ( Xoo ) on a 1/2 trypsin soybean agar (TSB) agar plate (BD Biosciences) at 28 ° C . Pseudomonas taiwanensis precultures were grown in an iron-limited medium (M9 basic medium supplemented with 1% casein amino acid, 1 mM MgSO 4 and 0.5% glycerol), and cultivated at 28 ° C. and 200 rpm In a 500 ml flask containing 100 ml of culture medium for 24 h. Xoo precultures were grown in 1/2 TSB medium at 28 ° C for 3 days. Mix Xoo with molten 1/2 agar medium and then pour into empty dishes. For biological analysis, Pseudomonas taiwanensis (10 9 CFU / ml) or filtered (0.22 μm) supernatant was injected into the wells of an Xoo mixed LB agar plate until the inhibition zone was characterized.

(2)藉由LC/MS比較螢綠素(m/z 1044)含量 (2) Comparison of chlorophyll (m / z 1044) content by LC / MS

培育1天之後,藉由在4500g下離心10min收集培養物上清液。經 由0.22μm過濾器使培養物上清液滅菌。藉由冷凍乾燥來乾燥10mL等分之各過濾上清液,且使其再懸浮於50%甲醇中。藉由高解析液相層析-質譜分析(LC/MS)(ESI-Orbitrap,由Metabolomics Core Facility,Academia Sinica,Taiwan執行)偵測代謝物之總數。測定峰值高度及面積以用於計算LC/MS分析中之螢綠素含量。 After 1 day of incubation, the culture supernatant was collected by centrifugation at 4500 g for 10 min. through The culture supernatant was sterilized by a 0.22 μm filter. A 10 mL aliquot of each filtered supernatant was dried by lyophilization and resuspended in 50% methanol. The total number of metabolites was detected by high-resolution liquid chromatography-mass spectrometry (LC / MS) (ESI-Orbitrap, performed by Metabolomics Core Facility, Academia Sinica, Taiwan). The peak height and area were measured to calculate the chlorophyll content in the LC / MS analysis.

(3)建構轉座子庫 (3) Constructing transposon libraries

EZ-Tn5轉座子突變誘發套組(KAN-2;Epicentre)用於製造無規突變體庫。根據製造商之說明書執行EZ-Tn5轉座子突變誘發。根據Choi等人(J Microbiol Methods 64:391-397,2006)中概述之方法製備台灣假單孢菌勝任細胞。為了篩選Tn5突變體庫,吾人採用台灣假單孢菌突變誘發庫與Xoo一起培育,提供了發現毒性相關基因之機會。藉由TAIL-PCR擴增插入位點之側接序列。由Sun等人(FEMS Microbiol Lett,226:145-150,2003)設計兩組無規引子及轉座子引子之兩端特異性區域。藉由PCR及定序進一步測定此研究之Tn5突變菌株。藉由UV光測定突變菌株(clpVpvdLpvdE)。將台灣假單孢菌clpVpvdLpvdE之核苷酸序列提交至Genbank資料庫,寄存編號分別為KM061430、KM036007及KM036029。最後,吾人使用南方墨點分析(Southern blot analysis)檢查Tn5插入突變體插入數目。藉由具有DIG標記PCR探針之南方墨點雜交來分析Tn5插入突變體之NcoI消化基因組DNA及EagI消化基因組DNA。具有康微素抗性基因之探針的南方分析用於證實插入數目。雜交之後,使用偵測套組(Roche)使得南方墨點顯色。 The EZ-Tn5 transposon mutation-inducing set (KAN-2; Epicentre) was used to make a random mutant library. EZ-Tn5 transposon mutation induction was performed according to the manufacturer's instructions. Pseudomonas taiwanese competent cells were prepared according to the methods outlined in Choi et al. (J Microbiol Methods 64: 391-397, 2006). In order to screen the Tn5 mutant library, we used the Taiwan Pseudomonas mutation-induced library to cultivate with Xoo, providing an opportunity to discover genes related to toxicity. Flanking sequences at the insertion site were amplified by TAIL-PCR. Sun et al. (FEMS Microbiol Lett, 226: 145-150, 2003) designed two sets of random primers and transposon primer-specific regions at both ends. The Tn5 mutant strains studied were further determined by PCR and sequencing. Mutant strains ( clpV , pvdL , pvdE ) were determined by UV light. The nucleotide sequences of clpV , pvdL, and pvdE of Pseudomonas taiwan were submitted to the Genbank database, and the registration numbers were KM061430, KM036007, and KM036029, respectively. Finally, we used Southern blot analysis to check the number of Tn5 insertion mutant insertions. NcoI-digested genomic DNA and EagI-digested genomic DNA of Tn5 insertion mutants were analyzed by Southern blot blotting with DIG-labeled PCR probes. Southern analysis of probes with a Kangxin protein was used to confirm the number of insertions. After hybridization, a Southern Ink Dot was developed using a detection kit (Roche).

為了監視clpV之下游基因表現,吾人藉由RT-PCR偵測WT及clpV突變體中之PT3445及yhfE基因表現。結果展示,clpV突變不影響下游基因表現。clpV突變體由廣泛宿主範圍載體pCPP30表現補充。藉由添加最終1mM異丙基-β-D-硫代半乳糖苷(IPTG)至鐵有限培養基中隔夜誘發含有clpV片段之pCPP30。 In order to monitor the downstream gene expression of clpV , we used RT-PCR to detect the PT3445 and yhfE gene expression in WT and clpV mutants. The results showed that clpV mutation did not affect downstream gene performance. The clpV mutant is complemented by a broad host range vector pCPP30. PCPP30 containing clpV fragments was induced overnight by adding the final 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to the iron-limited medium.

(4)分泌T6SS組分 (4) secretion of T6SS components

使用抗根癌農桿菌(anti-Agrobacterium tumefaciens)VgrG抗體,在培養物上清液中藉由西方墨點法偵測VgrG以確保T6SS活性。使用抗根癌農桿菌RpoA抗體偵測RNA聚合酶a-次單元RpoA,其用作西方墨點中之內參考物。兩種抗VgrG及抗RpoA抗體由Institute of Plant and Microbial Biology,Academia Sinica,Taiwan之Dr.Erh-Min Lai提供。在鐵有限培養基中培養台灣假單孢菌野生型及clpV突變體二十四小時,在600nm下(00600)生長至約0.8之光學密度。在4500g下離心10min之後,經由0.22μm Durapore聚偏二氟乙烯(PVDF)(最低蛋白結合)針筒過濾器使培養物上清液滅菌。在4℃下藉由添加三氯乙酸(TCA)至最終10% TCA濃度隔夜使無細胞培養物上清液蛋白(20ml)沈澱,且用冰冷丙酮洗滌集結粒兩次以移除殘餘TCA。使TCA沈澱分泌蛋白溶解於9.8M尿素溶液中。 Anti- Agrobacterium tumefaciens VgrG antibody was used to detect VgrG in the culture supernatant by Western blot method to ensure T6SS activity. An anti-Agrobacterium tumefaciens RpoA antibody was used to detect the RNA polymerase a-subunit RpoA, which was used as an internal reference in Western blots. Two anti-VgrG and anti-RpoA antibodies were provided by the Institute of Plant and Microbial Biology, Academia Sinica, Dr. Erh-Min Lai of Taiwan. The Pseudomonas taiwan wild type and clpV mutants were cultured in iron limited medium for twenty-four hours, and grown to an optical density of about 0.8 at 600 nm (00600). After centrifugation at 4500 g for 10 min, the culture supernatant was sterilized through a 0.22 μm Durapore polyvinylidene fluoride (PVDF) (minimum protein binding) syringe filter. Cell-free culture supernatant protein (20 ml) was precipitated overnight by adding trichloroacetic acid (TCA) to a final 10% TCA concentration at 4 ° C, and the pellets were washed twice with ice-cold acetone to remove residual TCA. The TCA precipitated secreted proteins were dissolved in a 9.8 M urea solution.

(5)MALDI-IMS (5) MALDI-IMS

藉由MALDI-IMS比較代謝物在競爭瓊脂盤表面上之分佈顯示台灣假單孢菌之野生型及突變體分泌之離子中的令人感興趣之差異。切除細菌群落之關注區且將其置放於玻璃載玻片上。使用50μm篩網,用具有沈積於樣本上之通用MALDI基質(Sigma-Aldrich)的薄層覆蓋具有令人感興趣之靶向樣本之載玻片。在IMS之前,在37℃下在培育箱中使基質覆蓋瓊脂樣本脫水隔夜。藉由Bruker Autoflex Speed MALDI-TOF/TOF MS分析樣本且收集資料。以正極反射型離子模式分析樣本,在200μm雷射間隔下篩選,其中獲取質量範圍設定為100Da至2000Da。使用標準肽校準混合物(肽校正標準206195,Bruker,1000Da至3200Da)及基質校準設備。使用Fleximaging 3.0軟體(Bruker)分析IMS資料。分子強度呈現為梯度顏色。 Comparing the distribution of metabolites on the surface of competing agar plates by MALDI-IMS shows interesting differences in the ions secreted by wild type and mutants of Pseudomonas taiwanensis. The area of interest of the bacterial community was excised and placed on a glass slide. A 50 μm sieve was used to cover a glass slide with a targeted sample of interest with a thin layer with a universal MALDI matrix (Sigma-Aldrich) deposited on the sample. Prior to IMS, the matrix-covered agar samples were dehydrated overnight in an incubator at 37 ° C. Samples were collected and data collected by Bruker Autoflex Speed MALDI-TOF / TOF MS. The samples were analyzed in a positive reflection ion mode and screened at a laser interval of 200 μm, where the acquisition mass range was set to 100 Da to 2000 Da. A standard peptide calibration mixture (Peptide Calibration Standard 206195, Bruker, 1000 Da to 3200 Da) and a matrix calibration device were used. Fleximaging 3.0 software (Bruker) was used to analyze IMS data. The molecular intensity appears as a gradient color.

(6)螢綠素之純化及測定 (6) Purification and determination of fluorescein

自Yin等人(Biosensors & bioelectronics 51,90-96(2014))修改螢綠素純化方法。在28℃及200rpm下,在鐵有限培養基中培育於250ml燒瓶之50ml台灣假單孢菌24h。藉由在4℃下在4,600g下離心15min收集培養物上清液,且經由0.22μm無菌低蛋白結合聚偏二氟乙烯(PVDF)膜濾器(Millex-GV;Millipore)過濾。螯合Cu-瓊脂糖凝膠管柱用於純化螢綠素。銅離子(Cu2+)用於自Ni-瓊脂糖高效能(GE)再裝填瓊脂糖。將5ml Ni-瓊脂糖裝載於0.8×4cm Poly-Prep層析管柱(Bio-Rad)中,且藉由重力法允許緩衝液流經。為了移除殘餘Ni2+,用5管柱體積緩衝液(0.02M Na2HPO4、0.5M NaCl及0.05M EDTA;pH 7.2)洗滌Ni-瓊脂糖凝膠管柱。隨後,藉由至少5管柱體積蒸餾水洗滌管柱以移除殘餘EDTA,且用0.5ml 1M CuSO4再裝填瓊脂糖。因此,用5管柱體積結合緩衝液(0.02M Na2HPO4、1M NaCl;pH 7.2)洗滌Cu-瓊脂糖。 The chlorophyll purification method was modified from Yin et al. (Biosensors & bioelectronics 51, 90-96 (2014)). 50 ml of Pseudomonas taiwanensis was cultivated in a 250 ml flask at 28 ° C and 200 rpm for 24 h in an iron-limited medium. The culture supernatant was collected by centrifugation at 4,600 g for 15 min at 4 ° C, and filtered through a 0.22 μm sterile low protein-bound polyvinylidene fluoride (PVDF) membrane filter (Millex-GV; Millipore). Chelated Cu-Sepharose gel column was used for purification of luciferin. Copper ions (Cu 2+ ) are used to refill agarose from Ni-Sepharose High Performance (GE). 5 ml of Ni-Sepharose was loaded on a 0.8 × 4 cm Poly-Prep chromatography column (Bio-Rad), and the buffer solution was allowed to flow through by gravity. To remove residual Ni 2+ , the Ni-Sepharose column was washed with 5 column volume buffers (0.02M Na 2 HPO 4 , 0.5M NaCl, and 0.05M EDTA; pH 7.2). Subsequently, the column was washed with at least 5 column volumes of distilled water to remove residual EDTA, and the agarose was refilled with 0.5 ml of 1M CuSO 4 . Therefore, Cu-sepharose was washed with 5 column volume binding buffer (0.02M Na 2 HPO 4 , 1M NaCl; pH 7.2).

以1:1之比率混合過濾培養物上清液與結合緩衝液。在Cu-瓊脂糖凝膠管柱中將20ml混合物裝載至經純化螢綠素或其他噬鐵素中。用5管柱體積結合緩衝液再次洗滌管柱。最後,藉由溶離緩衝液(0.02M Na2HPO4及1M NH4Cl;pH 7.2)溶離噬鐵素且藉由冷凍乾燥器乾燥。藉由具有RP-醯胺C16管柱(4.6×250mm,5μm;Sigma-Aldrich)之HPLC分析及MALDI-TOF MS檢查經純化化合物。螢光螢綠素之吸收最大值波長在407nm至412nm內顯而易見。此處,藉由UV吸收偵測器在200nm至500nm範圍內監測HPLC之層析。HPLC流動相之乙腈-水梯度為以1ml/min流速在10min內自50%至0%乙腈。每分鐘收集溶離份且藉由MALDI-TOF偵測。為了識別結構特徵,藉由ESI-Orbitrap(Academia Sinica之代謝組研究核心)測定m/z 1044峰值。 The filter culture supernatant and the binding buffer were mixed at a ratio of 1: 1. 20 ml of the mixture was loaded into a purified luciferin or other ferritin in a Cu-sepharose column. Wash the column again with 5 column volumes of binding buffer. Finally, ferritin was lysed by lysis buffers (0.02M Na 2 HPO 4 and 1M NH 4 Cl; pH 7.2) and dried by a freeze dryer. The purified compounds were checked by HPLC analysis with RP-amidamine C16 column (4.6 × 250 mm, 5 μm; Sigma-Aldrich) and MALDI-TOF MS. The maximum absorption wavelength of fluorescein is obvious in the range of 407nm to 412nm. Here, the HPLC chromatography is monitored by a UV absorption detector in the range of 200 nm to 500 nm. The acetonitrile-water gradient of the HPLC mobile phase was from 50% to 0% acetonitrile at a flow rate of 1 ml / min in 10 min. Dissolved fractions were collected every minute and detected by MALDI-TOF. To identify the structural features, the m / z 1044 peak was determined by ESI-Orbitrap, the core of the metabolomics research of Academia Sinica.

(7)抑制濃度(IC50)及致死劑量(LD50)分析 (7) Analysis of inhibitory concentration (IC 50 ) and lethal dose (LD 50 )

使經純化螢綠素溶解於1/2 TSB中且藉由0.22過濾器滅菌。將5.5mg/ml至0mg/ml之含純螢綠素之1/2 TSB培養基置放於含2ml 1/2 TSB 之管中。為了研究螢綠素對Xoo生長之影響,在28℃及200rpm下培育兩晚之後,分析600nm處下之吸光率及活細胞數目(cfu/ml)。三次執行分析且獲得一致結果。 Purified luciferin was dissolved in 1/2 TSB and sterilized by a 0.22 filter. 5.5 mg / ml to 0 mg / ml 1/2 TSB medium containing pure chlorophyll was placed in a tube containing 2 ml 1/2 TSB. In order to study the effect of luciferin on the growth of Xoo , the absorbance and the number of viable cells (cfu / ml) at 600 nm were analyzed after two night incubation at 28 ° C and 200 rpm. The analysis was performed three times with consistent results.

(8)CAS盤分析 (8) CAS disk analysis

鉻奧醇S(CAS)為偵測鐵移動之通用方法,其分析噬鐵素產生。為了製備100ml CAS染料,使60.5mg CAS粉末(Sigma)溶解於50ml蒸餾水中,且與10ml 1mM鐵溶液(無水FeCl3,Alfa Aesar)混合。隨後,將40ml 72.9mg HDTMA(Sigma)緩慢添加至含FeCl3之60ml CAS溶液中,且高壓處理以滅菌。CAS冷卻可手持之後,混合十分之一CAS溶液與LP瓊脂培養基且立即傾入盤中。 Chromol S (CAS) is a universal method for detecting iron movement, which analyzes ferritin production. To prepare 100 ml of CAS dye, 60.5 mg of CAS powder (Sigma) was dissolved in 50 ml of distilled water and mixed with 10 ml of a 1 mM iron solution (anhydrous FeCl 3 , Alfa Aesar). Subsequently, 40 ml of 72.9 mg of HDTMA (Sigma) was slowly added to 60 ml of the CAS solution containing FeCl 3 , and treated under high pressure to sterilize. After the CAS has cooled and can be held by hand, mix one-tenth CAS solution with LP agar medium and immediately pour into the dish.

CAS盤用於證實經純化螢綠素活性。將不同濃度經純化螢綠素注入CAS盤之孔(5mm)中。在28℃下培育盤6h或直至出現黃暈。 CAS plates are used to confirm purified chlorophyll activity. Purified luciferin at different concentrations was injected into the wells (5 mm) of the CAS disk. Incubate the dish at 28 ° C for 6h or until yellow halo appears.

(9)定量亞細胞螢綠素 (9) Quantitative subcellular luciferin

在鐵有限培養基中生長14h之後,自台灣假單孢菌之無細胞培養物上清液定量細胞外成熟螢綠素。藉由離心(6,000×g,3min)收集培養物上清液且藉由0.22μm孔徑過濾器過濾。為了分離周質與胞溶質部分,根據Imperi等人(Proteomics 9:1901-1915,2009)中概述之方法獲得球形質體。在PBS緩衝液(pH 7.4)中洗滌細胞集結粒(3×109細胞)三次。使細胞集結粒懸浮於1mL球形質體緩衝液(10mM Tris-HCl,pH 8.0,200mM MgCh,0.5mg/mL溶菌酶)中且在室溫下伴隨溫和振盪培育30min。培育之後,藉由離心(11,000×g,15min,4℃)收集周質部分。在PBS緩衝液(pH 7.4)中洗滌球形質體三次。使集結粒懸浮於1mL音波處理緩衝液(10mM Tris-HCl,pH 8.0,100mM NaCl)中且藉由音波處理溶解。離心(16,000×g,5min)之後,移除細胞殘渣以獲得細胞質部分。使用適當稀釋之稀釋緩衝液(100mM Tris-HCl),使用具有405nm/460nm之激發/發射波長之螢光盤讀取器(Victor 2,Perkin-Elmer), 測定成熟螢光螢綠素。針對細胞光學密度(OD600)標準化螢綠素值。 After 14 h growth in iron-limited medium, extracellular mature luciferin was quantified from the cell-free culture supernatant of Pseudomonas taiwanensis. The culture supernatant was collected by centrifugation (6,000 × g, 3 min) and filtered through a 0.22 μm pore size filter. To separate the periplasmic and cytosolic fractions, spherical plastids were obtained according to the method outlined in Imperi et al. (Proteomics 9: 1901-1915, 2009). Cell aggregates (3 × 10 9 cells) were washed three times in PBS buffer (pH 7.4). Cell aggregates were suspended in 1 mL of spherical plastid buffer (10 mM Tris-HCl, pH 8.0, 200 mM MgCh, 0.5 mg / mL lysozyme) and incubated for 30 min at room temperature with gentle shaking. After incubation, the periplasmic fraction was collected by centrifugation (11,000 × g, 15 min, 4 ° C). Spheroids were washed three times in PBS buffer (pH 7.4). The aggregated particles were suspended in 1 mL of sonication buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaCl) and dissolved by sonication. After centrifugation (16,000 × g, 5min), the cell residue was removed to obtain a cytoplasmic fraction. Mature fluorescent chlorophyll was measured using a suitably diluted dilution buffer (100 mM Tris-HCl) using a fluorescent disc reader (Victor 2, Perkin-Elmer) with an excitation / emission wavelength of 405 nm / 460 nm. The fluorescein value is normalized for cell optical density (OD600).

實例2:藉由台灣假單孢菌處理Xoo感染水稻葉 Example 2 : Treatment of Xoo infected rice leaves by Pseudomonas taiwanensis

山茶水稻栽培品種Tainung 67(稻L.)用於盆栽實驗。吾人藉由剪刀夾方法,用Xoo感染6週齡植物之葉子。感染之後,立即將台灣假單孢菌培養物上清液或台灣假單孢菌培養物噴灑至植物。第一次噴灑之後,在兩週時間段期間再噴灑植物三次。感染三週之後,經處理葉子比未經處理之對照葉子顯著更健康,該等未經處理之對照葉子為乾燥及黃色的。 Camellia rice cultivar Tainung 67 (rice L.) was used for pot experiments. We infected the leaves of 6-week-old plants with Xoo by means of scissors. Immediately after infection, the Pseudomonas taiwanensis culture supernatant or Pseudomonas taiwanensis culture was sprayed onto the plants. After the first spray, the plants were sprayed three more times over a two-week period. Three weeks after infection, the treated leaves were significantly healthier than the untreated control leaves, which were dry and yellow.

實例3:台灣假單孢菌之殺蟲活性 Example 3 : Insecticidal activity of Pseudomonas in Taiwan

吾人發現台灣假單孢菌為廣泛宿主範圍的昆蟲病原細菌,其展現對農業害蟲小菜蛾、甜菜夜蛾、斜紋夜蛾(Spodoptera litura)、粉紋夜蛾及黑腹果蠅(Drosophila melanogaster)之殺蟲活性。不同濃度(OD=0.5至2)野生型台灣假單孢菌之經口感染產生無顯著差異的昆蟲死亡率(92.7%、96.4%及94.5%)。TccC蛋白(毒素複合物(Tc)之組分)在台灣假單孢菌之殺蟲活性中發揮基本作用。台灣假單孢菌之△tccC突變菌株(其在TccC基因中具有基因剔除突變)甚至在高細菌劑量(OD=2.0)下僅誘發42.2%之小菜蛾死亡率。TccC蛋白斷裂成兩個片段,含有Rhs樣域之N端片段及含有Glt同向轉運體域及TraT域之C端片段,其可能分別有助於針對巨噬細胞之抗氧化應激活性及防禦。令人感興趣地,台灣假單孢菌中TccC之C端區之一級結構在病原體中獨特。藉由流動式細胞量測術證明TccC之C端片段之膜定位。台灣假單孢菌△tccC菌株之音波處理集結粒針對Sf9昆蟲細胞株及小菜蛾幼蟲之毒性比野生型低。吾人亦發現受台灣假單孢菌感染之Sf9及LD652Y-5d細胞株誘發凋亡細胞死亡。此外,藉由台灣假單孢菌之天然經口感染觸發宿主程式性細胞死亡相關基因JNK-2及卡斯蛋白酶-3之表現。 I have found that Pseudomonas taiwani is a wide host range of insect pathogenic bacteria, which has shown its effects on the agricultural pests diamondback moth, beet armyworm, Spodoptera litura , Spodoptera litura , and Drosophila melanogaster Insecticidal activity. Oral infection of wild-type Pseudomonas spp. At different concentrations (OD = 0.5 to 2) produced no significant differences in insect mortality (92.7%, 96.4%, and 94.5%). TccC protein (a component of the toxin complex (Tc)) plays a fundamental role in the insecticidal activity of Pseudomonas taiwanensis. The △ tccC mutant strain of Pseudomonas taiwani (which has a gene knockout mutation in the TccC gene) only induced a mortality of 42.2% of the diamondback moth at high bacterial doses (OD = 2.0). The TccC protein is broken into two fragments, including the N-terminal fragment of the Rhs-like domain and the C-terminal fragment containing the Glt cotransporter domain and the TraT domain, which may contribute to the activation of antioxidant stress and defense against macrophages, respectively. . Interestingly, the primary structure of the C-terminal region of TccC in Pseudomonas taiwanii is unique among pathogens. The membrane localization of the C-terminal fragment of TccC was demonstrated by flow cytometry. The sonic-treated aggregates of Pseudomonas △ tccC strain in Taiwan were less toxic to Sf9 insect cell lines and Plutella xylostella larvae than the wild type. We also found that Sf9 and LD652Y-5d cell lines infected with Pseudomonas taiwani induced apoptosis of apoptotic cells. In addition, the natural oral infection of Pseudomonas spp. In Taiwan triggered the expression of host programmed cell death related genes JNK-2 and Cassin-3.

台灣假單孢菌之TccC對小菜蛾之殺蟲活性Insecticidal activity of Pseudomonas Taiwanese TccC against Plutella xylostella

在先前研究中,來自台灣假單孢菌之TccC基因在大腸桿菌(E.coli)中過度表現,且重組TccC能夠增加果蠅幼蟲之死亡率。參見Liu等人,Journal of Agricultural and Food Chemistry 58:12343-12349(2010)。除黑腹果蠅之外,吾人發現台灣假單孢菌針對多種鱗翅目物種具有殺蟲活性,包括若干植物害蟲小菜蛾、甜菜夜蛾及粉紋夜蛾。 In previous studies, the TccC gene from Pseudomonas taiwanii was overexpressed in E. coli , and recombinant TccC could increase the mortality of Drosophila larvae. See Liu et al., Journal of Agricultural and Food Chemistry 58: 12343-12349 (2010). In addition to Drosophila melanogaster, we have found that Pseudomonas taiwan has insecticidal activity against a variety of Lepidoptera species, including several plant pests, diamondback moth, Spodoptera exigua, and Spodoptera litura.

吾人研究台灣假單孢菌TccC針對鱗翅目物種小菜蛾之活體內殺蟲活性。當細菌細胞達至生長停滯期(24h)時,TccC在台灣假單孢菌中之表現量最高(圖4A)。因此,吾人收集此階段之台灣假單孢菌細胞且測定其毒性。向小菜蛾幼蟲經口投與台灣假單孢菌細胞。處理組中之幼蟲展現較慢生長,且與對照組中之幼蟲相比為黑化、脫水且硬質的(圖4B)。 We studied the in vivo insecticidal activity of Pseudomonas taiwanensis TccC against the diamondback moth, Plutella xylostella. When the bacterial cells reached the growth stagnation period (24h), TccC showed the highest expression in Pseudomonas spp. (Figure 4A). Therefore, we collected Pseudomonas taiwanii cells at this stage and determined their toxicity. Oral administration of Plutella xylostella cells to Plutella xylostella larvae. The larvae in the treatment group exhibited slower growth and were melanized, dehydrated, and rigid compared to the larvae in the control group (Figure 4B).

吾人比較來自不同病原體之若干TccC樣蛋白之胺基酸序列,且發現其全部均具有N端保守RhsA樣域及C端高變片段。令人感興趣地,台灣假單孢菌之TccC在C端區中具有獨特鈉/麩胺酸同向轉運體樣域及TraT樣域。為了評估TccC蛋白之功能,吾人產生台灣假單孢菌之同基因tccC基因之基因剔除突變體,命名為△tccC。表1展示藉由野生型或△tccC台灣假單孢菌之全細胞或不同細胞部分經口投與之小菜蛾幼蟲的死亡率。經台灣假單孢菌△tccC菌株(OD=2.0)感染之小菜蛾幼蟲死亡率僅為42.4%,而經野生型台灣假單孢菌感染之小菜蛾幼蟲死亡率為94.5%(表1)。 We compared the amino acid sequences of several TccC-like proteins from different pathogens and found that all of them have N-terminally conserved RhsA-like domains and C-terminal hypervariable fragments. Interestingly, the TccC of Pseudomonas taiwan has a unique sodium / glutamic acid transporter-like domain and a TraT-like domain in the C-terminal region. In order to evaluate the function of the TccC protein, we produced a knockout mutant of the isogenic tccC gene of Pseudomonas taiwan , named ΔtccC . Table 1 shows the mortality of Plutella xylostella larvae orally administered by whole cells or different cell parts of Pseudomonas taiwanensis by wild type or ΔtccC . The mortality rate of Plutella xylostella larvae infected with Pseudomonas △ tccC strain (OD = 2.0) was only 42.4%, and that of Plutella xylostella larvae infected with Pseudomonas taiwanensis was 94.5% (Table 1).

a 給三齡期之健康幼蟲喂台灣假單孢菌野生型、△tccC突變菌株及其各種蛋白部分。 a third instar larvae to the health of the Taiwan Pseudomonas feed wild type, △ tccC mutant strains and their various protein fractions.

b 死亡率為幼蟲死亡百分比。n為處理組之樣本大小。資料在第5天收集。 b Mortality is the percentage of larval deaths. n is the sample size of the treatment group. Information was collected on the 5th day.

c 雙尾斯登氏t-測試用於闡明統計顯著性。各處理重複三次。 c Two-tailed Stern's t-test is used to elucidate statistical significance. Each treatment was repeated three times.

d 類似於b,n為陰性對照PBS處理組之樣本大小。 d is similar to b , and n is the sample size of the negative control PBS-treated group.

e 攝入劑量:50μl OD=0.5、1、2個細胞/0.5*1cm2植物塊。 e Intake dose: 50 μl OD = 0.5, 1, 2 cells / 0.5 * 1 cm 2 plant pieces.

f 攝入劑量:粗提取物含有300ng蛋白。 f Intake dose: The crude extract contains 300ng of protein.

吾人進一步製備台灣假單孢菌之不同細胞部分,且測試其對小菜蛾幼蟲之影響。大於50%之經野生型台灣假單孢菌之細胞溶解物、不溶性溶解物(細胞膜及細胞壁集結粒)及細胞外上清液感染之小菜蛾幼蟲在5天喂飼期結束時死亡(表1)。另外,經台灣假單孢菌△tccC之細胞溶解物及不溶性集結粒感染之小菜蛾幼蟲的死亡率低於經野生型溶解物感染之小菜蛾幼蟲的死亡率(表1)。此等結果表明台灣假單孢菌之殺蟲活性可能至少部分歸因於TccC。 I further prepared different cell parts of Pseudomonas taiwanii and tested their effects on Plutella xylostella larvae. More than 50% of P. xylostella larvae infected with wild-type Pseudomonas taiwani cell lysates, insoluble lysates (cell membrane and cell wall aggregates) and extracellular supernatant died at the end of the 5-day feeding period (Table 1 ). In addition, the mortality of P. xylostella larvae infected with Pseudomonas △ tccC and insoluble aggregates was lower than that of P. xylostella larvae infected with wild-type lysates (Table 1). These results indicate that the insecticidal activity of Pseudomonas taiwan may be at least partially attributed to TccC.

用毒素、細菌或病毒感染鱗翅目幼蟲導致受損腸上皮細胞中出現頂部突起及突起破裂。因此,吾人執行組織學分析來評定台灣假單孢菌感染對小菜蛾之腸道之影響。小菜蛾幼蟲之中腸超微結構展示經口 感染台灣假單孢菌對腸細胞具有強烈影響。感染台灣假單孢菌48h之後,誘發小菜蛾腸中之腸上皮細胞之頂部突起、異常微絨毛及細胞溶解,表明台灣假單孢菌感染對中腸上皮細胞導致嚴重損傷,其在體內恆定過程中無法修復且最終導致宿主死亡。類似地,攝入100ng毒素複合物(Tc)/cm2食物之小菜蛾幼蟲之超微結構切片在含諸多囊泡樣結構之腸中展示柱狀細胞。相比而言,攝入△tccC突變體僅展示異常微絨毛而無任何頂部突起或細胞溶解。 Infection of Lepidoptera larvae with toxins, bacteria, or viruses causes apical processes and ruptures of processes in damaged intestinal epithelial cells. Therefore, we performed a histological analysis to assess the effect of Pseudomonas Taiwan infection on the intestinal tract of Plutella xylostella. The ultrastructure of midgut of Plutella xylostella larvae showed that oral infection with Pseudomonas taiwan has a strong effect on intestinal cells. 48 hours after infection with Pseudomonas taiwanensis, induced top protrusions, abnormal microvilli, and cytolysis of intestinal epithelial cells in the intestine of Plutella xylostella, suggesting that Pseudomonas taiwanensis infection caused severe damage to midgut epithelial cells and its constant process in vivo It cannot be repaired and eventually causes the host to die. Similarly, ultrastructural sections of Plutella xylostella larvae that ingested 100ng of toxin complex (Tc) / cm 2 food displayed columnar cells in the intestine containing many vesicle-like structures. In contrast, the ingested ΔtccC mutant displayed only abnormal microvilli without any top protrusions or cytolysis.

對腸之損傷可誘發幹細胞增殖且分化而替代受損細胞,產生大量形狀比對照組大之杯狀細胞。吾人觀察到,與非感染或野生型台灣假單孢菌感染小菜蛾相比,經台灣假單孢菌△tccC經口感染之小菜蛾在中腸系統中產生大量杯狀細胞,表明僅△tccC之感染而非野生型可誘發中腸系統中受損細胞之分化及諸多杯狀物之形成。此表明台灣假單孢菌△tccC之毒性低於野生型菌株之毒性,且中腸上皮細胞可在過程中修復。 Injury to the intestine can induce stem cell proliferation and differentiation to replace the damaged cells, resulting in a large number of goblet cells that are larger than the control group. It observed that, compared to non-infected or wild-type Pseudomonas infection Taiwan Plutella xylostella, dried cinerea tccC orally infected Plutella goblet cells produce large amounts of the intestinal system in Taiwan Pseudomonas, showed only tccC The infection rather than the wild type can induce the differentiation of damaged cells in the midgut system and the formation of many cups. This indicates that the toxicity of △ tccC of Pseudomonas taiwanii is lower than that of wild-type strains, and midgut epithelial cells can be repaired in the process.

細菌定量及組織學檢查進一步證實台灣假單孢菌對小菜蛾之中腸上皮細胞之定殖及侵入。經口感染48h之後,台灣假單孢菌△tccC之細菌計數低於小菜蛾中腸中野生型菌株之細菌計數。另外,經口感染48h之後,中腸上皮細胞受野生型台灣假單孢菌嚴重破壞。 Bacterial quantification and histological examination further confirmed the colonization and invasion of P. xylostella into the midgut epithelial cells of Plutella xylostella. After 48 hours of oral infection, the bacterial count of Pseudomonas △ tccC in Taiwan was lower than that of the wild type strain in the midgut of Plutella xylostella. In addition, 48 hours after oral infection, midgut epithelial cells were severely damaged by wild-type Pseudomonas taiwanensis.

藉由用不同台灣假單孢菌細胞部分處理Sf9昆蟲細胞進一步證實TccC之殺蟲活性。參見圖5。暴露於野生型台灣假單孢菌之完整細胞(活台灣假單孢菌)、細胞溶解物(總蛋白)、可溶性溶解物(胞溶質蛋白)及不溶性溶解物(細胞壁及細胞膜)之Sf9昆蟲細胞的存活率顯著低於暴露於PBS緩衝液之Sf9昆蟲細胞存活率。另一方面,暴露於台灣假單孢菌△tccC之完整細胞或細胞壁集結粒之Sf9昆蟲細胞的存活率與暴露於PBS緩衝液之Sf9昆蟲細胞存活率無顯著差異,僅暴露於台灣假單孢菌△tccC之細胞溶解物或可溶性溶解物之Sf9昆蟲細胞存活率顯著降低。 由於台灣假單孢菌△tccC確實不表現TccC,因此一些其他毒性因子很可能存在於台灣假單孢菌△tccC之細胞溶解物中。此外,活性吞噬作用可見於Sf9活細胞中,活體內凋亡期間之特有現象,但在活體外培養物中不常見。Sf9細胞為吞噬細胞的且含有異常高數目之吞噬體,尤其在葡萄糖耗竭後。在早期感染階段中(培育1h之後),RFP標記台灣假單孢菌經Sf9細胞吞噬。培育3h之後,與非感染細胞不溶解相比,觀察到經台灣假單孢菌感染之Sf9細胞之溶解。 The insecticidal activity of TccC was further confirmed by partially treating Sf9 insect cells with different Pseudomonas cells from Taiwan. See Figure 5. Sf9 insect cells exposed to intact cells (live Pseudomonas taiwan), cell lysates (total protein), soluble lysates (cytosolic proteins), and insoluble lysates (cell wall and cell membrane) of wild-type The survival rate was significantly lower than that of Sf9 insect cells exposed to PBS buffer. On the other hand, the survival rate of Sf9 insect cells exposed to intact cells of Pseudomonas △ tccC or cell wall aggregated granules was not significantly different from the survival rate of Sf9 insect cells exposed to PBS buffer, only to Pseudomonas taiwanensis The survival rate of Sf9 insect cells in cell lysates or soluble lysates of bacteria △ tccC was significantly reduced. Because Taiwan pseudomonas △ tccC does not express TccC, some other toxicity factors are likely to be present in the cell lysate of Taiwan pseudomonas △ tccC . In addition, active phagocytosis can be seen in Sf9 living cells, a unique phenomenon during in vivo apoptosis, but is not common in in vitro cultures. Sf9 cells are phagocytic and contain an abnormally high number of phagosomes, especially after glucose depletion. In the early infection stage (after 1 h incubation), RFP-labeled Pseudomonas taiwanensis was phagocytosed by Sf9 cells. After 3 h of incubation, lysis of Sf9 cells infected with Pseudomonas taiwanese was observed as compared to non-infected cells that did not lyse.

藉由台灣假單孢菌之TccC誘發凋亡細胞死亡Apoptotic cell death induced by TccC of Pseudomonas taiwanensis

為了確定台灣假單孢菌感染在鱗翅目Sf-9及LD-5d細胞中是否誘發凋亡,吾人使用磷脂結合蛋白V-FITC染色使凋亡細胞及DAPI染色來測定總細胞數目。用台灣假單孢菌感染10h之後,在鱗翅目Sf-9及LD-5d細胞中偵測凋亡,且觀察到比非感染對照中顯著較高死亡率。此外,藉由台灣假單孢菌感染觸發小菜蛾幼蟲之腸上皮細胞之JNK路徑。除JNK路徑之外,吾人亦檢查卡斯蛋白酶基因之表現,其亦可誘發凋亡細胞死亡。經口感染台灣假單孢菌48h之後,中腸細胞中斷裂卡斯蛋白酶-3之表現量增加。經台灣假單孢菌△tccC感染之小菜蛾幼蟲中JNK-2及斷裂卡斯蛋白酶-3之表現量低於台灣假單孢菌之野生型菌株中之表現量,表明TccC可能誘發凋亡且在小菜蛾幼蟲腸上皮細胞之細胞死亡中發揮重要作用。 To determine whether Pseudomonas taiwanensis infection induced apoptosis in Lepidoptera Sf-9 and LD-5d cells, we used phospholipid binding protein V-FITC staining to stain apoptotic cells and DAPI to determine the total cell number. After 10 hours of infection with Pseudomonas taiwanii, apoptosis was detected in Sf-9 and LD-5d cells of Lepidoptera, and a significantly higher mortality rate was observed than in non-infected controls. In addition, the JNK pathway of the intestinal epithelial cells of Plutella xylostella larvae was triggered by Pseudomonas spp. Infection in Taiwan. In addition to the JNK pathway, we also examined the performance of the Cassin gene, which can also induce apoptosis of apoptotic cells. 48 hours after oral infection with Pseudomonas taiwanii, the expression of cleaved Cassin-3 in midgut cells increased. The expression levels of JNK-2 and cleaved Cassin -3 in Plutella xylostella larvae infected with Pseudomonas △ tccC in Taiwan were lower than those in wild-type strains of Pseudomonas taiwanii, indicating that TccC may induce apoptosis and Plays an important role in the cell death of intestinal epithelial cells of the diamondback moth larvae.

TccC對台灣假單孢菌之抗氧化活性之影響Effect of TccC on the Antioxidant Activity of Pseudomonas taiwanensis

藉由完整腸上皮屏障及宿主免疫防禦系統,保護健康昆蟲之消化道以免細菌破壞。吾人分析台灣假單孢菌菌株之蛋白酶及抗氧化活性以評估其針對昆蟲腸免疫系統之抗性。在細菌生長之生長停滯期,台灣假單孢菌分泌大量蛋白酶且展示高抗氧化活性。台灣假單孢菌△tccC之抗氧化活性顯著低於野生型台灣假單孢菌之抗氧化活性,表明台灣假單孢菌之抗氧化活性可能受TccC直接或間接調節。 Protects the digestive tract of healthy insects from bacterial destruction with a complete intestinal epithelial barrier and host immune defense system. We analyzed the protease and antioxidant activities of Pseudomonas strains in Taiwan to assess their resistance to the intestinal immune system of insects. During the stagnant period of bacterial growth, Pseudomonas taiwan secretes a large amount of proteases and exhibits high antioxidant activity. The anti-oxidant activity of △ tccC of Pseudomonas taiwanii was significantly lower than that of wild-type Pseudomonas taiwanii , indicating that the anti-oxidant activity of Pseudomonas taiwanii may be directly or indirectly regulated by TccC.

為了證實TccC與抗氧化活性有關,使野生型及△tccC台灣假單孢菌暴露於不同濃度之過氧化氫且測定細菌計數。結果展示野生型台灣假單孢菌之存活率比△tccC高,證實TccC在針對ROS之細菌細胞之保護中亦發揮作用。ROS在高濃度H2O2處理下誘發tccC突變體之較大損傷。台灣假單孢菌TccC蛋白在其C端中含有鈉/麩胺酸同向轉運體Glts樣域,其可能用於麩胺酸輸送。由於L-麩胺酸可轉化成麩胱甘肽,因此TccC可能在針對ROS侵襲之防禦中發揮作用,且維持台灣假單孢菌中之細胞內氧化還原電位。吾人隨後測定台灣假單孢菌是否具有降解過氧化氫(H2O2)之能力。吾人發現1mM H2O2在用野生型台灣假單孢菌培育2min之後快速降解。相比而言,當用tccC突變體培育時,花費15min完全分解。總之,吾人之結果表明,野生型台灣假單孢菌具有較高H2O2解毒活性,且因此可比tccC突變體更有效地保護自身免受由宿主免疫反應產生的ROS侵襲。 To confirm that TccC is related to antioxidant activity, wild-type and ΔtccC Pseudomonas Taiwanese were exposed to different concentrations of hydrogen peroxide and the bacterial counts were determined. The results show that the survival rate of wild-type Pseudomonas taiwanii is higher than △ tccC , confirming that TccC also plays a role in the protection of bacterial cells against ROS. ROS induced greater damage to the tccC mutant under high concentration H 2 O 2 treatment. The Pseudomonas taiwanensis TccC protein contains a sodium / glutamate isoporter Glts-like domain in its C-terminus, which may be used for glutamate transport. Since L-glutamic acid can be converted into glutathione, TccC may play a role in defense against ROS invasion and maintain the intracellular redox potential in Pseudomonas taiwanensis. We then determined whether Pseudomonas taiwan has the ability to degrade hydrogen peroxide (H 2 O 2 ). We found that 1 mM H 2 O 2 was rapidly degraded after 2 min incubation with wild-type Pseudomonas taiwanensis. In contrast, when incubated with the tccC mutant, it took 15 min to completely decompose. In summary, our results indicate that wild-type Pseudomonas taiwanii has a higher H 2 O 2 detoxification activity, and therefore can protect itself from ROS from the host immune response more effectively than tccC mutants.

TccC之抗噬菌活性Anti-phage activity of TccC

為了評估TccC之抗噬菌活性,吾人執行吞噬作用分析,其中野生型及△tccC台灣假單孢菌細胞螢光標記有CFSE,且隨後與小鼠巨噬細胞一起培育。與螢光標記台灣假單孢菌△tccC一起培育30min之巨噬細胞展示峰值位置朝向較高螢光強度之位移,表明經吞噬△tccC之量大於經吞噬野生型台灣假單孢菌之量。為了證明散佈圖分析之研究成果,計算經吞噬台灣假單孢菌之百分比。與△tccC細胞相比,小鼠巨噬細胞吞噬更少野生型細胞,表明野生型台灣假單孢菌具有抗吞噬活性,其可部分歸因於TccC。吾人亦分析台灣假單孢菌野生型及△tccC對小鼠巨噬細胞之細胞毒性,且發現小鼠巨噬細胞之存活率在野生型存在下與在△tccC存在下不同,表明台灣假單孢菌對小鼠巨噬細胞不具有細胞毒素作用。 In order to evaluate the anti-phage activity of TccC, we performed a phagocytosis analysis, in which wild-type and ΔtccC Pseudomonas Taiwanese cells were fluorescently labeled with CFSE, and then incubated with mouse macrophages. Macrophages incubated for 30 minutes with fluorescently labeled Pseudomonas taiwantccC showed a shift in peak position toward higher fluorescence intensity, indicating that the amount of phagocytosed △ tccC was greater than that of wild type Pseudomonas taiwanensis . In order to demonstrate the results of the scatter plot analysis, the percentage of Pseudomonas spp. That had been engulfed was calculated. Compared with △ tccC cells, mouse macrophages phagocytose fewer wild-type cells, indicating that wild-type Pseudomonas taiwanensis has anti-phagocytotic activity, which can be attributed in part to TccC. We also analyzed the cytotoxicity of Pseudomonas taiwan wild type and △ tccC on mouse macrophages, and found that the survival rate of mouse macrophages was different in the presence of wild type and in the presence of △ tccC , indicating that Taiwan pseudomonas Spores have no cytotoxic effect on mouse macrophages.

TccC活體內處理及位置TccC in vivo processing and location

基於Pfam域預測,預測TccC具有RhsA域(11-673)、Rhs重複相關核心(600-680)、鈉/麩胺酸同向轉運體樣(726-825)及TraT補充抗性樣域(736-781)。另外,預測C端區三個跨膜區(718-742、744-758、760-778)。執行西方墨點法分析以測定TccC蛋白在台灣假單孢菌中之亞細胞定位。根據圖6中概述方法製備三個細胞部分。出人意料地,在總細胞蛋白部分中偵測到兩個蛋白帶(約70KD及約40KD帶),表示TccC蛋白之處理形式。在可溶性蛋白部分中,僅偵測到約70kD帶,而在含有細胞壁及膜蛋白之不溶性集結粒部分中,僅偵測到經處理約40kD帶。此表明當插入台灣假單孢菌細胞膜中時,TccC蛋白經處理。 Based on Pfam domain prediction, TccC is predicted to have RhsA domain (11-673), Rhs repeat-related core (600-680), sodium / glutamate isoporter-like (726-825), and TraT supplementary resistance-like domain (736 -781). In addition, three transmembrane regions (718-742, 744-758, 760-778) are predicted for the C-terminal region. Western blot analysis was performed to determine the subcellular localization of TccC protein in Pseudomonas taiwanensis. Three cell fractions were prepared according to the method outlined in FIG. 6. Surprisingly, two protein bands (about 70KD and about 40KD bands) were detected in the total cellular protein fraction, indicating the processed form of the TccC protein. Only about 70 kD bands were detected in the soluble protein portion, and only about 40 kD bands were detected in the insoluble agglomerate portion containing cell wall and membrane proteins. This indicates that the TccC protein was processed when inserted into the cell membrane of Pseudomonas taiwanensis.

吾人已觀察到,重組TccC蛋白亦在大腸桿菌表現系統中經類似處理。為了進一步表徵斷裂過程,將具有6×His-標記物之TccC選殖入廣泛宿主範圍載體pCPP30中且其在台灣假單孢菌及大腸桿菌(BL21)中過度表現。使用鎳離子管柱純化標記His之TccC蛋白。西方墨點分析展示具有類似分子量之TccC蛋白之處理形式自大腸桿菌與台灣假單孢菌經純化。此結果表明,TccC在大腸桿菌與台灣假單孢菌中具有類似斷裂位點。 We have observed that the recombinant TccC protein is similarly treated in the E. coli expression system. To further characterize the fragmentation process, TccC with a 6 × His-tag was cloned into a wide host range vector pCPP30 and it was over-expressed in Pseudomonas taiwan and E. coli (BL21). TccC protein labeled with His was purified using a nickel ion column. Western blot analysis showed that processed forms of TccC proteins with similar molecular weights were purified from E. coli and Pseudomonas taiwanensis. This result indicates that TccC has similar break sites in E. coli and Pseudomonas taiwanensis.

為了測試TccC是否真正整合入細胞膜中,用FITC標記TccC以藉由TccC-FITC抗體染色來追蹤外膜部分。流動式細胞量測術分析展示,台灣假單孢菌之細胞表面上之TccC螢光信號比非染色對照具有顯著較高密度。相比而言,在tccC突變體中未偵測到顯著螢光密度。 To test whether TccC is truly integrated into the cell membrane, TccC was labeled with FITC to track the outer membrane portion by staining with TccC-FITC antibodies. Flow cytometry analysis showed that the TccC fluorescence signal on the cell surface of Pseudomonas taiwanii had a significantly higher density than the unstained control. In contrast, no significant fluorescence density was detected in the tccC mutant.

材料及方法Materials and methods (1)細菌菌株、培養條件及抗生素 (1) Bacterial strains, culture conditions and antibiotics

台灣假單孢菌BCRC 17751用作昆蟲病原物種。大腸桿菌DH5α用於所有建構實驗。大腸桿菌S17-1用於與台灣假單孢菌之兩親配對,且大腸桿菌BL21用於表現重組蛋白。台灣假單孢菌及大腸桿菌在魯利亞-貝爾塔尼(Luria-Bertani;LB)培養液中或瓊脂盤上生長。台灣假單孢 菌培養物在30℃下生長且大腸桿菌培養物在37℃下生長。抗生素在以下濃度下施用:立複黴素(rifampicin)(34μg/ml)、安比西林(ampicillin)(100μg/ml)及大觀黴素(spectinomycin)(100μg/ml)用於台灣假單孢菌野生型經培養之培養基;及康微素(kanamycin)(30μg/ml)、四環素(20μg/ml)分別用於台灣假單孢菌突變菌株及過度表現菌株;康微素(50μg/ml)、安比西林(100μg/ml)及四環素(20μg/ml)用於大腸桿菌菌株。 Pseudomonas taiwanensis BCRC 17751 is used as an insect pathogenic species. E. coli DH5α was used in all construction experiments. E. coli S17-1 was used to pair with the amphiphiles of Pseudomonas taiwan, and E. coli BL21 was used to express recombinant proteins. Pseudomonas taiwanii and E. coli were grown in Luria-Bertani (LB) culture medium or on agar plates. Pseudomonas taiwanensis The bacteria culture was grown at 30 ° C and the E. coli culture was grown at 37 ° C. Antibiotics were administered at the following concentrations: rifampicin (34 μg / ml), ampicillin (100 μg / ml), and specinomycin (100 μg / ml) for Pseudomonas in Taiwan wild Type cultured medium; Kanamycin (30 μg / ml) and tetracycline (20 μg / ml) are used for Pseudomonas mutans strains and over-expressing strains in Taiwan; Kang Weisu (50 μg / ml), Ambi Xilin (100 μg / ml) and tetracycline (20 μg / ml) were used for E. coli strains.

(2)細胞培養 (2) Cell culture

鱗翅目昆蟲草地黏蟲Sf9細胞株與舞毒蛾(Lymantria dispar)IPLB LD-652Y-5d細胞株由Dr.C.H.Wang(國立台灣大學之昆蟲學系(Department of Entomology,National Taiwan University))提供。自IPLB LD-652Y[47]次選殖舞毒蛾(gypsy moth/Lymantria dispar)細胞株IPLB LD-652Y-5d。其在27℃下在補充有10%胎牛血清(FBS)及1%青黴素/鏈黴素/麩醯胺酸(PSG)(Invitrogen)之Sf-900 II SFM(Gibco)培養基中生長。 Lepidopteran grassland armyworm Sf9 cell line and Lymantria dispar IPLB LD-652Y-5d cell line were provided by Dr. CH Wang (Department of Entomology, National Taiwan University). From the IPLB LD-652Y [47] sub-selection of the gypsy moth / Lymantria dispar cell line IPLB LD-652Y-5d. It was grown at 27 ° C in Sf-900 II SFM (Gibco) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin / glutamine (PSG) (Invitrogen).

(3)台灣假單孢菌△tccC基因剔除突變體之建構 (3) Construction of △ tccC gene knockout mutant of Pseudomonas taiwanensis

藉由含有插入之康微素抗性盒之tccC片段之自殺載體pEX100T的雙重再組合來建構命名為△tccC之台灣假單孢菌之tccC(GenBank資料庫寄存編號HQ260745)基因剔除突變體。藉由將1345-bp康微素抗性盒插入含有tccC編碼序列之852-bp片段中來產生tccC-kan-tccC片段。將tccC-kan-tccC片段選殖入pEX100T自殺載體中,且隨後轉型至大腸桿菌S17-1中,從而與野生型台灣假單孢菌結合。在含有5%蔗糖、30μg/ml康微素、34μg/ml立複黴素及100μg/ml大觀黴素之LB盤上選擇雙重再組合tccC突變菌株。藉由PCR及定序來證實所得△tccC突變體。 By pEX100T double suicide vector containing the fragment was inserted into the tccC Kang-resistant micro-cassette of the recombined to construct named △ tccC Taiwan tccC of Pseudomonas (GenBank Database Accession No. HQ260745) knockout mutant. The tccC-kan-tccC fragment was generated by inserting a 1345-bp conmicin resistance cassette into a 852-bp fragment containing the tccC coding sequence. The tccC-kan-tccC fragment was cloned into the pEX100T suicide vector and then transformed into E. coli S17-1 to bind to wild-type Pseudomonas taiwanensis. A double recombination tccC mutant strain was selected on LB discs containing 5% sucrose, 30 μg / ml Kangweisuin, 34 μg / ml ritomycin, and 100 μg / ml spectinomycin. The resulting ΔtccC mutant was confirmed by PCR and sequencing.

(3)感染實驗及有效蛋白部分之生物分析 (3) Infection experiment and biological analysis of effective protein

藉由天然經口感染執行幼蟲之細菌感染之生物分析。使台灣假單孢菌生長24小時至生長停滯期且收集。隨後,在5ml PBS(pH 7.4)中洗 滌細胞集結粒三次且使其再懸浮於PBS中,調節至不同濃度(OD)。將不同濃度細菌(50μl)施加至0.5×1cm2植物塊表面,該等植物塊用於喂植物娥小菜蛾之幼蟲且在25℃下培育。經口感染之後,在第5天觀察各經感染幼蟲且計算死亡率。健康第三齡期小菜蛾幼蟲由台灣農業化學物質及有毒物質研究研究所(Taiwan Agricultural Chemicals and Toxic Substances Research Institute)提供。為了測定導致小菜蛾死亡之蛋白部分,培養台灣假單抱菌24小時。藉由離心(在4,600g,4℃下15min)收穫細胞培養物,且單獨收集上清液及細胞集結粒。對於培養物上清液,經由0.22μm PVDF過濾器(Millipore)過濾分泌蛋白且使用Vivaspin 20濃縮器(10kDa MWCO,GE Healthcare)濃縮。用PBS洗滌收穫細胞集結粒兩次,且使其再懸浮於含蛋白酶抑制劑之PBS中且藉由音波處理溶解(細胞溶解物)。藉由離心(在26,000g,4℃下30min)將細胞溶解物分離成不溶性溶解物及可溶性溶解物,且藉由0.22μm PVDF過濾器過濾可溶性溶解物。用PBS洗滌不溶性溶解物兩次且使其再懸浮於PBS中。對於來自台灣假單孢菌之蛋白部分之毒性分析,溶解於10μl PBS中之300ng蛋白用於昆蟲幼蟲處理。藉由Pierce 660nm蛋白分析法(Pierce)定量蛋白質提取物。 Biological analysis of bacterial infection of larvae was performed by natural oral infection. Pseudomonas taiwanii was grown for 24 hours to a growth stagnation period and collected. Subsequently, the cell aggregates were washed three times in 5 ml of PBS (pH 7.4) and resuspended in PBS, and adjusted to different concentrations (OD). Bacteria of different concentrations (50 μl) were applied to the surface of 0.5 × 1 cm 2 plant pieces, which were used to feed the larvae of the plant Plutella xylostella and cultivated at 25 ° C. After oral infection, each infected larva was observed on day 5 and mortality was calculated. Healthy third-instar diamondback moth larvae were provided by the Taiwan Agricultural Chemicals and Toxic Substances Research Institute. To determine the portion of the protein that caused the death of Plutella xylostella, Pseudomonas taiwanii was cultured for 24 hours. Cell cultures were harvested by centrifugation (15 minutes at 4,600 g, 4 ° C), and the supernatant and cell aggregates were collected separately. For the culture supernatant, the secreted protein was filtered through a 0.22 μm PVDF filter (Millipore) and concentrated using a Vivaspin 20 concentrator (10 kDa MWCO, GE Healthcare). The harvested cell aggregates were washed twice with PBS and resuspended in PBS containing protease inhibitors and lysed (cell lysate) by sonication. The cell lysate was separated into insoluble lysate and soluble lysate by centrifugation (at 26,000 g, 4 min at 4 ° C), and the soluble lysate was filtered through a 0.22 μm PVDF filter. The insoluble lysate was washed twice with PBS and resuspended in PBS. For the toxicity analysis of the protein portion of Pseudomonas taiwanii, 300 ng of protein dissolved in 10 μl of PBS was used for insect larvae treatment. Protein extracts were quantified by Pierce 660nm protein analysis (Pierce).

(4)細胞存活分析 (4) Cell survival analysis

為了研究台灣假單孢菌對昆蟲細胞之影響,藉由比色XTT分析測定草地黏蟲Sf9細胞之增殖。對於細胞毒性分析,以5,000個/孔將Sf9細胞接種於補充有10μg/ml台灣假單孢菌之各種部分蛋白之96孔培養盤中,或在無抗生素培養基中每細胞添加1000Pt感染倍率(MOI)。處理72h之後,藉由細胞增殖分析套組(XTT)(Biological Industries)定量細胞增殖。 In order to study the effect of Pseudomonas taiwanensis on insect cells, the colorimetric XTT analysis was used to determine the proliferation of Sf9 cells of the grassland armyworm. For cytotoxicity analysis, Sf9 cells were seeded at 5,000 cells / well in 96-well culture plates supplemented with 10 μg / ml of various partial proteins of Pseudomonas taiwanensis, or 1000 Pt infection rates were added per cell in antibiotic-free medium ( MOI). After 72 h of treatment, cell proliferation was quantified by the Cell Proliferation Assay Kit (XTT) (Biological Industries).

(5)凋亡分析 (5) Apoptosis analysis

藉由磷脂結合蛋白V-FITC分析偵測細胞早期凋亡。藉由計數在螢 光顯微鏡下可見的磷脂結合蛋白V陽性細胞測定人類或昆蟲細胞之凋亡百分比。在24孔盤之孔上,以10μg/ml用台灣假單孢菌之蛋白部分或台灣假單孢菌(MOI=1000)培育細胞(5,000個細胞/孔)72h。處理72h之後,在PBS中洗滌細胞兩次,且根據製造商之說明書使用ApoAlert磷脂結合蛋白V-FITC套組(BD)偵測。用4',6-二甲脒基-2-苯基吲哚二乳酸鹽(DAPI)染色細胞核中之DNA 5min。最終,在PBS中洗滌染色細胞兩次,用4%多聚甲醛固定10分鐘且隨後在螢光顯微鏡(Zeiss Axiovert 100M,Carl Zeiss,Germany)下觀察。計數磷脂結合蛋白V陽性細胞且識別為台灣假單孢菌誘發早期凋亡細胞。 Early cell apoptosis was detected by phospholipid binding protein V-FITC analysis. By counting on the firefly Phospholipid-binding protein V-positive cells visible under a light microscope were used to determine the percentage of apoptosis in human or insect cells. Cells (5,000 cells / well) were cultured on the wells of a 24-well plate at 10 μg / ml with the protein fraction of Pseudomonas taiwanensis or Pseudomonas taiwanensis (MOI = 1000) for 72 h. After 72 h of treatment, cells were washed twice in PBS and detected using ApoAlert phospholipid binding protein V-FITC kit (BD) according to the manufacturer's instructions. DNA in the nucleus was stained with 4 ', 6-dimethylformyl-2-phenylindole dilactate (DAPI) for 5 min. Finally, the stained cells were washed twice in PBS, fixed with 4% paraformaldehyde for 10 minutes and then observed under a fluorescent microscope (Zeiss Axiovert 100M, Carl Zeiss, Germany). Phospholipid-binding protein V-positive cells were counted and identified as early apoptotic cells induced by Pseudomonas taiwanensis.

(6)切片及HE、革蘭氏、免疫組織化學染色 (6) Sections and HE, Gram, immunohistochemical staining

細菌經口感染48h之後,在10%緩衝福馬林(pH 7.0)中固定第三齡期幼蟲至少48h。固定之後,將幼蟲遞送至國立台灣大學之病理部實驗室以用於切片。藉由蘇木精-伊紅、革蘭氏(Gram's)或免疫組織化學染色分析組織切片。使用抗JNK-2[N1C3](GTX105523,Genetex;與家蠶(Bombyx mori,NP_001103396)之c-Jun NH2封端激酶80%[276/398]序列一致性)及抗卡斯蛋白酶-3 p17(GTX123678,Genetex;與家蠶(AAW79564)之卡斯蛋白酶36%[46/129]序列一致性)抗體執行免疫組織化學(IHC)染色,之後用二胺基聯苯胺(DAB)顯色且用來自國立台灣大學醫院實驗動物中心(Laboratory Animal Center of National Taiwan University Hospital)之蘇木精對比染色。 After 48 hours of oral infection, the third instar larvae were fixed in 10% buffered formalin (pH 7.0) for at least 48 hours. After fixation, the larvae were delivered to the Department of Pathology, National Taiwan University for sectioning. Tissue sections were analyzed by hematoxylin-eosin, Gram's or immunohistochemical staining. Uses anti-JNK-2 [N1C3] (GTX105523, Genetex; 80% [276/398] sequence identity with c-Jun NH2 capping kinase of Bombyx mori (NP_001103396)) and anti-cassin-3 p17 (GTX123678 , Genetex; immunohistochemical (IHC) staining with a 36% [46/129] sequence identity of Cassin protein from silkworm (AAW79564), followed by color development with diaminobenzidine (DAB) and from National Taiwan Hematoxylin contrast staining at the Laboratory Animal Center of National Taiwan University Hospital.

(7)純化TccC (7) Purified TccC

將全長TccC-His6融合片段選殖入廣泛宿主範圍Pcpp30載體中且轉型至大腸桿菌(BL21)及台灣假單孢菌中。台灣假單抱菌及大腸桿菌生長至生長停滯期(24h)之後,藉由His SpinTrap管柱(GE Healthcare)純化過度表現之TccC-His6融合蛋白,且藉由使用抗TccC抗體之西方墨點法顯示結果。 The full-length TccC-His 6 fusion fragment was cloned into a wide host range Pcpp30 vector and transformed into E. coli (BL21) and Pseudomonas taiwanensis. After Pseudomonas spp. And E. coli were grown to the growth stagnant period (24h), the over-expressed TccC-His 6 fusion protein was purified by His SpinTrap column (GE Healthcare), and by western blots using anti-TccC antibodies Method to display the results.

(8)分析TccC位置 (8) Analysis of TccC position

對於SDS PAGE,使來自台灣假單孢菌之20μg不同細胞部分蛋白質溶解於含SDS之加樣緩衝液中,且隨後施用於凝膠電泳。電泳之後,以40mA將蛋白質轉移至硝化纖維膜歷時12h。使用針對自大腸桿菌BL21表現純化之台灣假單孢菌TccC全長重組蛋白產生之兔多株抗體,用特異性抗TccC抗體偵測TccC。一級抗體結合之後,用辣根過氧化酶偶合的抗兔二級抗體結合及化學發光偵測試劑(Pierce)使顏色顯色。 For SDS PAGE, 20 μg of different cell fraction proteins from Pseudomonas taiwan were dissolved in a loading buffer containing SDS, and then applied to gel electrophoresis. After electrophoresis, the protein was transferred to the nitrocellulose membrane at 12 mA for 12 h. Multiple anti-TccC antibodies were used to detect TccC using rabbit antibodies raised against the full-length recombinant Pseudomonas TccC protein purified from E. coli BL21 expression. After the binding of the primary antibody, the horseradish peroxidase-conjugated anti-rabbit secondary antibody binding and chemiluminescence detection reagent (Pierce) were used to develop the color.

流動式細胞量測術用於測定TccC之膜定位。使台灣假單抱菌之野生型及△TccC突變菌株生長隔夜且在生長停滯期(24h)收集。調節培養物109CFU/ml,且隨後離心100l經調節細菌,從而收集集結粒。在4℃下用PBS洗滌細菌集結粒三次且其再懸浮於含1% BSA之200μl PBS中。在冰上將多株抗TccC抗體(1/100稀釋)添加至細菌懸浮液中1h。用PBS再洗滌細菌三次,且在冰上用山羊FITC結合抗兔IgG二級抗體(1/100稀釋)(Jackson Immunoresearch)染色1h。染色之後,洗滌細菌三次且使其再懸浮於1ml PBS中且藉由流動式細胞量測術分析。使用Summit 5.2軟體(Beckman Coulter),藉由MoFlo XDP Cell Sorter(Beckman Coulter)執行流動式細胞量測術。 Flow cytometry is used to determine the membrane localization of TccC. The wild-type and △ TccC mutant strains of Pseudomonas taiwanii were grown overnight and collected during the growth stagnation period (24h). The culture was adjusted to 10 9 CFU / ml, and then 100 l of the regulated bacteria were centrifuged to collect aggregate particles. The bacterial aggregates were washed three times with PBS at 4 ° C and resuspended in 200 μl PBS containing 1% BSA. Multiple anti-TccC antibodies (1/100 dilution) were added to the bacterial suspension on ice for 1 h. Bacteria were washed three more times with PBS and stained with goat FITC-conjugated anti-rabbit IgG secondary antibody (1/100 dilution) (Jackson Immunoresearch) on ice for 1 h. After staining, the bacteria were washed three times and resuspended in 1 ml PBS and analyzed by flow cytometry. Flow cytometry was performed using Summit 5.2 software (Beckman Coulter) and MoFlo XDP Cell Sorter (Beckman Coulter).

(9)吞噬作用分析 (9) Analysis of phagocytosis

在生長停滯期早期收集台灣假單孢菌細胞,且用PBS洗滌兩次且使其再懸浮於PBS中至OD=1(4×109個細胞)。將一毫升再懸浮細胞添加至CFSE中(最終濃度5μM),且在30℃下在暗處培育30min。用PBS洗滌細胞三次且在螢光顯微鏡下觀察。對於吞噬作用分析,在37℃下在暗處將CSFE標記台灣假單孢菌細胞添加至巨噬細胞(MOI=1000)持續30min,且隨後用PBS洗滌三次。分別藉由流動式細胞量測術及螢光顯微鏡量測吞噬作用之定量及觀察結果。藉由Cytomics FC500(Beckman Coulter),使用CXP軟體(Beckman Coulter)執行流動式細胞量測術。收集一萬個細胞用於分析。非感染巨噬細胞用作陰性對照。 Pseudomonas taiwan cells were collected early in the growth arrest phase, washed twice with PBS and resuspended in PBS to OD = 1 (4 × 10 9 cells). One milliliter of resuspended cells was added to CFSE (final concentration 5 μM) and incubated at 30 ° C. for 30 min in the dark. Cells were washed three times with PBS and observed under a fluorescent microscope. For phagocytosis analysis, CSFE-labeled Pseudomonas taiwanensis cells were added to macrophages (MOI = 1000) for 30 min at 37 ° C in the dark, and then washed three times with PBS. Quantification and observation of phagocytosis were measured by flow cytometry and fluorescence microscopy, respectively. Flow Cytometry was performed with Cytomics FC500 (Beckman Coulter) and CXP software (Beckman Coulter). Ten thousand cells were collected for analysis. Non-infected macrophages were used as negative controls.

(10)台灣假單孢菌之定量H2O2分析及增殖分析 (10) Quantitative H 2 O 2 analysis and proliferation analysis of Pseudomonas taiwanensis

收集生長至生長停滯期(24h)之台灣假單孢菌細胞,在PBS中洗滌三次且以109個細胞/ml使其再懸浮於PBS中,且隨後用1M H2O2培育。在使用PeroX-Oquant定量過氧化物分析套組(Pierce)處理之後的不同時間點偵測剩餘H2O2之濃度。如先前所描述觀測羥基在台灣假單孢菌中之增殖作用。使台灣假單孢菌在LB培養液中生長24h,且隨後用不同濃度H2O2培育3h。藉由計數群落形成單位測定增殖。 Pseudomonas taiwani cells grown to growth arrest phase (24h) were collected, washed three times in PBS and resuspended in PBS at 10 9 cells / ml, and then incubated with 1M H 2 O 2 . The remaining H 2 O 2 concentration was detected at different time points after treatment with the PeroX-Oquant quantitative peroxide analysis kit (Pierce). The proliferation of hydroxyl groups in Pseudomonas taiwanensis was observed as described previously. Pseudomonas taiwanii was grown in LB culture for 24 h, and then incubated with H 2 O 2 at different concentrations for 3 h. Proliferation was determined by counting colony forming units.

其他實施例 Other embodiments

本說明書中揭示之所有特徵可以任何組合形式組合。本說明書中揭示之各特徵可經用於相同、等效或類似目的之替代性特徵置換。因此,除非另外明確說明,否則所揭示之各特徵僅為一系列通用等效或類似特徵之一個實例。 All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Therefore, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

自上述實施方式,熟習此項技術者可易於確定所描述實施例之基本特徵,且在不背離其精神及範疇之情況下可對實施例作出各種變化及修改以使其適應各種用途及條件。因此,其他實施例亦在申請專利範圍內。 From the above embodiments, those skilled in the art can easily determine the basic characteristics of the described embodiment, and can make various changes and modifications to the embodiment to adapt it to various uses and conditions without departing from its spirit and scope. Therefore, other embodiments are also within the scope of patent application.

<110> 中央研究院 施明哲 劉嚞睿 楊玉良 陳文仁 <110> Academia Sinica Shi Mingzhe Liu Yirui Yang Yuliang Chen Wenren

<120> 使用台灣假單孢菌(Pseudomonas taiwanensis)控制昆蟲及微生物之組合物及方法 <120> Composition and method for controlling insects and microorganisms using Pseudomonas taiwanensis

<130> A218936/189885 <130> A218936 / 189885

<140> 104118951 <140> 104118951

<141> 2015-06-11 <141> 2015-06-11

<150> US 62/010,776 <150> US 62 / 010,776

<151> 2014-06-11 <151> 2014-06-11

<160> 4 <160> 4

<170> PatentIn version 3.5 <170> PatentIn version 3.5

<210> 1 <210> 1

<211> 3522 <211> 3522

<212> 去氧核醣核酸 <212> DNA

<213> 台灣假單孢菌 <213> Pseudomonas taiwanensis

<400> 1 <400> 1

<210> 2 <210> 2

<211> 980 <211> 980

<212> 蛋白質 <212> Protein

<213> 台灣假單孢菌 <213> Pseudomonas taiwanensis

<400> 2 <400> 2

<210> 3 <210> 3

<211> 1008 <211> 1008

<212> 去氧核醣核酸 <212> DNA

<213> 台灣假單抱菌 <213> Taiwan pseudomonas

<400> 3 <400> 3

<210> 4 <210> 4

<211> 335 <211> 335

<212> 蛋白質 <212> Protein

<213> 台灣假單孢菌 <213> Pseudomonas taiwanensis

<400> 4 <400> 4

Claims (12)

一種產生抑制微生物生長之組合物的方法,該方法包含在養分有限培養基中培養台灣假單孢菌(Pseudomonas taiwanensis)菌株以獲得培養液及收集該培養液,從而產生該組合物,其中該微生物選自由以下組成之群:水稻黃單孢菌水稻致病變種(Xanthomonas oryzae pv.Oryzae)、似膠黏孢炭疽刺盤孢菌(Colletotrichum gloeosporioides)、辣椒疫黴菌(Phytophthora capsici)、稻熱病菌(Pyricularia oryzae)、立枯絲核菌(Rhizoctonia solani)、蘭花萎凋病菌(Fusarium oxysporum f sp cattleyae)、表皮葡萄球菌(Staphylococcus epidermidis)、金黃色葡萄球菌(Staphylococcus aureus)或白色念珠菌(Candida albican),其中該培養基為鐵有限培養基。A method for producing a composition for inhibiting the growth of microorganisms, the method comprising culturing a Pseudomonas taiwanensis strain in a nutrient-limited medium to obtain a culture solution and collecting the culture solution to produce the composition, wherein the microorganism is selected Free of the following groups: Xanthomonas oryzae pv. Oryzae , Colletotrichum gloeosporioides , Phytophthora capsici , Pyricularia oryzae ), Rhizoctonia solani , Fusarium oxysporum f sp cattleyae , Staphylococcus epidermidis , Staphylococcus aureus or Candida albican , of which The medium is an iron limited medium. 如請求項1之方法,其中該培養基為補充有酪蛋白胺基酸、MgSO4及甘油之M9基本培養基。The method of claim 1, wherein the medium is a M9 basic medium supplemented with casein amino acid, MgSO 4 and glycerol. 如請求項2之方法,其進一步包含自該培養液移除細胞以獲得無細胞上清液及收集該無細胞上清液。The method of claim 2, further comprising removing cells from the culture medium to obtain a cell-free supernatant and collecting the cell-free supernatant. 如請求項1之方法,其中該台灣假單孢菌菌株具有食品工業研究所寄存編號BCRC 17751。The method of claim 1, wherein the strain of Pseudomonas taiwan has the deposit number BCRC 17751 of the Institute of Food Industry. 如請求項1之方法,其中該台灣假單孢菌菌株具有功能喪失rpoS突變。The method of claim 1, wherein the Pseudomonas taiwan strain has a loss of function rpoS mutation. 一種抑制微生物生長之方法,該方法包含使該微生物與如請求項1至5中任一項之方法所產生之組合物接觸,其中該微生物選自由以下組成之群:水稻黃單孢菌水稻致病變種、似膠黏孢炭疽刺盤孢菌、辣椒疫黴菌、稻熱病菌、立枯絲核菌、蘭花萎凋病菌、表皮葡萄球菌、金黃色葡萄球菌或白色念珠菌。A method for inhibiting the growth of a microorganism comprising contacting the microorganism with a composition produced by the method of any one of claims 1 to 5, wherein the microorganism is selected from the group consisting of: Xanthomonas oryzae pv. Oryzae Pathological species, Anthracnose-like anthracnose, Phytophthora capsici, Pyricularia oryzae, Rhizoctonia solani, Fusarium oxysporum, Staphylococcus epidermidis, Staphylococcus aureus or Candida albicans. 一種治療或降低水稻細菌性枯葉病風險之方法,該方法包含向有需要之水稻植物施用如請求項1至5中任一項之方法所產生之組合物。A method for treating or reducing the risk of bacterial bacterial leaf blight of rice, the method comprising applying to a rice plant in need a composition produced by the method of any one of claims 1 to 5. 一種抑制微生物生長之方法,該方法包含:使該微生物與具有結構Q-DSer-Lys-OHHis-aDThr-Ser-cOHOrn之經分離螢綠素接觸,其中Q為發色團且該微生物選自由以下組成之群:水稻黃單孢菌水稻致病變種、似膠黏孢炭疽刺盤孢菌、辣椒疫黴菌、稻熱病菌、立枯絲核菌、蘭花萎凋病菌、表皮葡萄球菌、金黃色葡萄球菌或白色念珠菌。A method for inhibiting the growth of a microorganism, the method comprising: contacting the microorganism with an isolated chlorophyll having the structure Q-DSer-Lys-OHHis-aDThr-Ser-cOHOrn, wherein Q is a chromophore and the microorganism is selected from the group consisting of Composition group: Xanthomonas oryzae pv. Oryzae, Gliocladium anthracis, Phytophthora capsici, Pyricularia oryzae, Rhizoctonia solani, Fusarium oxysporum, Staphylococcus epidermidis, Staphylococcus aureus Or Candida albicans. 如請求項8之方法,其中該微生物為水稻黃單孢菌水稻致病變種。The method according to claim 8, wherein the microorganism is a rice pathogenic strain of Xanthomonas oryzae. 一種抑制昆蟲生長之方法,該方法包含使該昆蟲與含有台灣假單孢菌菌株、台灣假單孢菌細胞溶解物或台灣假單孢菌TccC多肽之組合物接觸,其中該昆蟲為為小菜蛾(Plutella xylostella)、甜菜夜蛾(Spodoptera exigua)或粉紋夜蛾(Trichoplusia ni)。A method for inhibiting the growth of an insect, the method comprising contacting the insect with a composition containing Pseudomonas taiwanii strain, Pseudomonas taiwan cell lysate, or Pseudomonas taiwanensis TccC polypeptide, wherein the insect is Plutella xylostella ( Plutella xylostella ), Spodoptera exigua , or Trichoplusia ni . 如請求項10之方法,其中該細胞溶解物為全細胞溶解物或可溶性溶解物。The method of claim 10, wherein the cell lysate is a whole cell lysate or a soluble lysate. 如請求項11之方法,其中該台灣假單孢菌菌株在富含養分之培養基中經培養,且該細胞溶解物自富含養分之培養基中培養之台灣假單孢菌菌株獲得。The method according to claim 11, wherein the Pseudomonas taiwan strain is cultured in a nutrient-rich medium, and the cell lysate is obtained from the Pseudomonas taiwan strain cultured in a nutrient-rich medium.
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